MEDICAL    .SCHOOL 
LIISMAIIO' 


ELEMENTS  OF 


Clinical  Bacteriology 


FOR 


PHYSICIANS  AND  STUDENTS 


BY 

DR.  ERNST  .LEVY 

Professor  in  the  University  of  Strasburg  i.  E. 

AND 

DR.  FELIX  KLEMPERER 

Private  Decent  in  the  University  of  Strasburg  i.  E. 


SECOND  ENLARGED  AND  REVISED  EDITION 

AUTHORIZED  TRANSLATION  BY 

AUGUSTUS  A.  ESHNER,  M.D. 

Professor  of  Clinical  Medicine  in  the  Philadelphia  Polyclinic ;  Physician  to  the 
Philadelphia  Hospital ;  etc. 


PHILADELPHIA 

W.   B.   SAUNDERS 
925  WALNUT  Street 


Copyright,  1900,  by  w.  b.  Saunders. 


Press  of 

•W%B.*|Al»IDEKS,VP^II^.t)4iPblM 


TRANSLATOR'S  NOTE. 


Works  on  bacteriology,  on  pathology,  and  on  clinical 
diagnosis  are  not  wanting  in  any  language,  but  we  know 
of  only  one  on  clinical  bacteriology,  and  as  a  consideration 
of  the  subject  from  this  aspect  must  appeal  to  a  large  body 
of  the  profession,  it  was  thought  a  service  might  be  ren- 
dered by  a  translation  of  this  admirable  publication,  whose 
authors  are  well  and  favorably  known  for  their  original 
work  in  both  clinical  medicine  and  bacteriology.  The 
general  practitioner  can  scarcely  be  expected  to  be  a 
trained  and  practised  bacteriologist,  but  he  must  have  a 
working  familiarity  with  the  subject  of  bacteriology  in  order 
that  he  may  possess  clear  conceptions  as  to  the  etiology  of 
disease  and  the  nature  of  the  resultant  morbid  processes, 
leading  to  a  rational  application  of  measures  and  methods 
of  prophylaxis  and  treatment.  To  these  ends  it  is  hoped 
the  present  publication  will  contribute.  The  work  of 
translation  is  coupled  with  especial  and  personal  pleasure 
from  the  fact  that  both  junior  author  and  translator 
participated  simultaneously  in  the  work  in  bacteriology 
in  the  Hygienic  Institute  at  Berlin  in  the  spring  of  1890. 

It  has  been  thought  advantageous  to  add  illustrations  to 
the  English  version.  A.  A.  E. 

Philadelphia,  February,  igoo. 


4^5 


Digitized  by  the  Internet  Archive 

in  2007  with  funding  from 

IVIicrosoft  Corporation 


http://www.archive.org/details/elementsofclinicOOIevyrich 


PREFACE  TO  THE  SECOND  EDITION. 


Scientific  activity  in  the  domain  of  clinical  bacteriology 
has  been  not  less  pronounced  than  fruitful  during  the 
period  that  has  elapsed  since  the  appearance  of  the  first 
edition  of  this  book.  By  including  the  numerous  results 
of  recent  investigation  the  size  of  the  present  volume  has 
been  considerably  increased.  Chapters  have  been  added 
on  Plague  and  Botulism,  and  those  on  Immunity,  Diph- 
theria, Typhoid  Fever,  Actinomycosis,  Examination  of  Air 
and  of  Water,  and  others,  have  been  radically  revised ;  and 
in  all  other  sections  numerous  changes  and  additions  have 
been  made. 

We  believe  the  work  adapted  to  the  present  position  of 
bacteriologic  knowledge,  and  hope  that  this  edition  may 
have  the  same  friendly  reception  accorded  the  first  edition. 

The  Authors. 


CONTENTS 


PART  L 

,  PAGE 

I.  Morphology  and  Biology  of  Bacteria 17 

II.  Infection 33 

III.  Immunity,  Immunization,  and  Cure 49 

IV.  Methods  of  Culture  and  of  Examination 74 


PART  n. 

INFLAMMATION   AND    SUPPURATION. 

Morphology  of  the  Causative  Agents  of  Inflammation I15 

The  Pathogenic  Properties  of  the  Causative  Agents  of  Inflammation  with 

Relation  to  Animals 123 

The  Occurrence  of  the  Causative  Agents  of  Inflammation  and  Suppura- 
tion in  Healthy  Persons  and  Outside  the  Body 126 

The  Occurrence  of  the  Causative  Agents  of  Inflammation  in  Disease  .    .  127 

Cutaneous  Suppuration 127 

Erysipelas 129 

Phlebitis  and  Lymphangitis 132 

Inflammations  of  the  Nose  and  Throat 133 

Angina      134 

Otitis  Media 135 

Meningitis ' . 136 

Bronchitis 138 

Pleuritis 138 

Pneumonia 142 

Endocarditis 148 

Pericarditis 150 

Myocarditis 15 1 

Peritonitis 151 

Cholecystitis  and  Cholangitis .' 154 

Abscess  of  the  Liver 155 

Nephritis -157 

Perinephritis 158 

Pyelonephritis 159 

13 


14  CONTENTS. 

PAGE 

Inflammatory  Diseases  of  the  Eye i6o 

Pyemia  and  Septicemia l6i 

Puerperal  Fever 163 

Osteomyelitis 164 

Pyocyaneous  General  Infection 165 


PARTm. 

SPECIFIC  DISEASES  OF  BACTERIAL  ORIGIN. 

Typhoid  Fever 166 

Asiatic  Cholera 181 

Cholera  Nostras  and  Summer  Diarrhea 198 

Plague 200 

Diphtheria 207 

Tetanus 230 

Botulism 245 

Tuberculosis 251 

Fowl-tuberculosis 275 

Pseudo- tuberculosis 276 

Leprosy 278 

Influenza 281 

Anthrax 287 

Glanders 300 

Malignant  Edema 305 

Proteus-infection      308 

Gonorrhea 311 

Syphilis 314 

Hydrophobia  (Lyssa  ;  Rabies) 321 

Smallpox  (Variola)  . ' "325 

Acute  Exanthemata 328 

Whooping-cough  (Pertussis) 330 

Articular  Rheumatism 331 

Relapsing  Fever 332 


PART  IV. 

I.  MYCOSES     (INFECTIONS     WITH     FILAMENTOUS     AND 

BUDDING  FUNGI). 

Morphology  and  Biology  of  the  Filamentous  and  the  Budding  Fungi     .  336 

Pathogenic  Activity  of  Filamentous  and  Budding  Fungi  for  Animals  .    .  339 

Diseases  in  Human  Beings  Induced  by  Filamentous  and  Budding  Fungi  341 

Actinomycosis 354 

Pathogenic  Streptothrices 362 

Pathogenic   Yeasts 363 


CONTENTS.  15 

PAGE 

II.  INFECTIONS  WITH  TliE  LOWEST  FORMS  OF 

ANIMAL  LIFE. 

Dysentery  (Amebic  Enteritis)  and  Tropical  Abscess  of  the  Liver  .    .    .  364 

Malaria 372 

Leydenia  Gemmipara  Schaudinn 387 


APPENDIX. 

I.  BACTERIOLOGIC    EXAMINATION    OF    SOIL,    AIR, 
AND    WATER. 

Soif 389 

Air     ... 392 

Water 396 


THE  BACTERIA  PRINCIPALLY  FOUND  IN  SOIL,  AIR, 
AND  WATER. 

I.   Bacilli 401 

II.   Micrococci 405 

III.   Vibrios 407 


II.  DISINFECTION. 

Disinfecting  Agents 411 

Disinfection  of  the  Hands 418 

Disinfection  of  Mucous  Membranes 419 

Disinfection  of  Instruments  and  Dressings 419 

Disinfection  of  Feces  and  Cesspools 419 

Disinfection  of  Bath-water,  Wash-water,  etc 420 

Disinfection  of  Urine      421 

Disinfection  of  Sputum 421 

Disinfection  of  Body-clothing  and  Bed-linen 422 

Disinfection  of  Beds  and  Clothing 423 

Disinfection  of  Articles  of  Food 423 

Disinfection  of  the  Sick-room 423 

Disinfection  of  Ships,  Vehicles,  Railway-cars,  etc 425 


Index 427 


I        \ 


)p  0  . 


425 


Clinical  Bacteriology, 


PART  I 


L  MORPHOLOGY  AND  BIOLOGY  OF  BACEB3UA. 

The  minute  forms  of  life  constituting  in  largest  part  the 
exciting  agents  of  the  infectious  diseases  occupy  the  lowest 
level  in  the  vegetable  kingdom.  They  are  designated  bac- 
teria or  fission-fungi.  The  latter  term  is,  however,  not  well 
chosen.  The  botanic  conception  of  fungi  includes  those 
lower  forms  of  vegetable  life  that  are  without  the  coloring- 
matter  of  leaves  (chlorophyl)  or  similar  coloring-matters 
(chromophyls).  There  are  not  a  few  bacteria,  however, 
that  are  capable  of  generating  such  coloring-matters,  and, 
thus,  also  of  decomposing  carbon  dioxid  and  its  combi- 
nations, and  utilizing  the  products  of  this  activity.  There 
is,  thus,  no  justification  for  the  term  fission-fungi,  and  it  is, 
therefore,  preferable  to  designate  the  minute  forms  of  life 
under  consideration  as  bacteria. 

There  is  as  yet  no  strictly  scientific,  natural  classification 
of  bacteria.  For  the  present  the  adoption  of  an  artificial 
system,  based  upon  such  physical  features  as  the  size,  the 
shape,  and  the  arrangement  of  the  bacteria,  must  suffice 
(F.  Cohn).  (See  Figs,  i,  2,  3.)  Three  main  divisions  are 
recognized:  (i)  Spheric  bacteria  (cocci,  micrococci,  coc- 
caceae)  ;  these  are  spheric  cells.  (2)  Rod-shaped  bacteria 
(bacilli,  bacteriaceae) ;  these  are  rod-like,  cylindric  cells. 
(3)  Spiral  bacteria  (vibrios,  spirilla,  spirillaceae) ;  these 
are  twisted  in  both  the  horizontal  and  the  vertical  direc- 
tion, and  are,  thus,  comparable  to  the  windings  of  a  screw 
or  of  a  corkscrew. 

2  17 


18 


CLINICAL  BACTERIOLOGY. 


The  individual  bacterial  cells  exhibit  at  times  a  homo- 
geneous translucent,  at  other  times  a  granular  protoplas- 
mic, structure  that,  on  the  whole,  possesses  the  properties 
of  all  other  protoplasmic  structure,  but  that  is  not,  as  in 
the  cells  of  the  higher  orders  of  plants  it  is,  differentiable 


0|?i 


\^ 


i^  fj  /< 


lO         11 


Fig.  I.— Various  forms  of  bacteria:  i  and  2,  Round  and  oval  micrococci ;  3,  diplo- 
cocci ;  4,  tetracocci,  or  tetrads;  5,  streptococci;  6,  bacilli;  7,  bacilli  in  chains,  the 
lower  showing  spore-formation ;  8,  bacilli  showing  spores,  forming  drumsticks  and 
Clostridia  ;  9  and  10,  spirilla;  11,  spirochetse  (McFarland). 


Fig.  2. — Diagram  illustrating  the  morphology  of  the  spirilla:  a,  ^,  c,  Spirilla;  d,  <?,, 
spirochetse  (McFarland). 


-^ 


/O 


Fig.  3.— Diagram  illustrating  the  morphology  of  the  bacilli :  a,  b,  c,  Various  forms 
of  bacilli ;  d,  <?,  bacilli  with  flagella  ;  f,  chain  of  bacilli,  individuals  distinct ;  ^,  chain 
of  bacilli,  individuals  not  separated  (McFarland). 


into  a  cell-nucleus  and  a  cell-body.  Nevertheless,  some 
observers  consider  the  so-called  central  body  in  certain 
large  bacteria  as  the  virtual  nucleus,  and  believe  the  plasma 
to  have  a  honeycomb  arrangement — a  view  that  has  recently 
been  put  forth  also  with  regard  to  the  large  species  of 
spirilla.    According  to  others,  the  interior  of  the  bacterium 


MORPHOLOGY  AND  BIOLOGY  OF  BACTERIA.     19 

consists  of  a  lining  layer  of  protoplasm,  and  a  loculated 
space  for  the  cell-fluid.  Not  rarely  the  protoplasm  of  the 
bacteria  contains  granules  that  stain  deeply  with  aniline 
dyes.  The  significance  of  these  so-called  Babes-Ernst 
bodies  has  not  been  established  ;  they  have  been  looked 
upon  as  the  progenitors  of  spores,  as  nuclear  structures,  as 
degeneration-products,  and,  finally,  as  reserve  substances. 

The  ground-substance  of  the  bacterial  cell  is  surrounded 
by  a  cell-capsule  or  cell-membrane  constituted  of  albuminoid 
material,  in  rare  instances  of  cellulose  and  of  varying 
amounts  of  a  carbohydrate  that  stains  blue  with  iodin. 
When  the  outer  layers  of  this  mem- 
brane possess  the  property  of  swelling 
up  greatly  and  becoming  viscid  and 
gelatinous,  an  appearance  is  created  as 
if  the  bacterium  possessed  a  special 
capsule^  and  under  such  circumstances 
it  is  customary,  in  view  of  the  physi- 

1  ^     ,  1       r  ,.  P'g-  4— Encapsulated 

cal  appearance,  to  speak  oi  an  encap-  bacteria. 

sulated  bacterium.      (Fig.    4.)      This 

mucoid  transformation  of  the  capsule  of  the  cell  occurs  in 
the  case  of  pathogenic  bacteria  usually  only  in  the  animal 
body,  and  but  seldom  in  artificial  culture-media. 

The  length  of  bacteria  varies  greatly  from  one  to  several 
micromillimeters  (twenty  and  more),  while  their  thickness 
is  generally  less  than  one  micromillimeter. 

Bacteria  are  in  part  motile,  in  part  nonmotile.  When 
motility  is  present,  it  is  dependent  upon  special  motor 
organs — so-called  flagella  (Fig.  5) — which  are  connected 
with  the  body  of  the  cell  in  varying  number  and  arrange- 
ment. These  represent  protoplasmic  structures  of  extreme 
attenuation,  which  arise  directly  from  the  cell -membrane, 
and  do  not  extend  into  the  actual  ground-substance.  The 
following  varieties  of  motile  bacteria  may  be  distinguished  : 

1.  Monotrocha,  with  a  single  flagellum  at  the  pole  of 
the  cell. 

2.  Amphitrocha,  with  a  single  flagellum  at  each  pole. 

3.  Lophotrocha,  with  a  cluster  of  flagella  at  one  pole. 

4.  Peritrocha,  with  a  varying  number  of  flagella  sur- 
rounding the  body  of  the  bacterium. 

The  shape  and  length  of  these  flagella  are  extremely 
variable  in  the  different  varieties  of  motile  bacteria.  The 
movement  itself  is  forward,  and  is  always  combined  with  a 


20  CLINICAL  BACTERIOLOGY. 

certain  amount  of  rotation.  It  is  most  marked  in  quite 
young  cultures,  but  is  in  greater  degree  dependent  upon  the 
variety  of  bacterium,  the  culture-medium,  and  the  tempera- 
ture. Many  species  of  bacteria  possess  spontaneous  move- 
ment throughout  their  whole  life  ;  others  only  during 
certain  phases,  losing  it,  for  instance,  preceding  spore- 
formation. 

The  multiplication  of  bacteria  takes  place  usually  by 
means  of  fission  in  pairs.  The  time  in  which  this  division 
occurs  varies  for  different  varieties.  In  the  case  of  some 
species,  if  all  of  the  extrinsic  conditions  (culture-medium. 


®  ®  @ 


Fig.  5. — Bacteria  showing:  flagella  :  i,  Bacilli  of  typhoid  fever;  2,  bacilli  coli  com- 
munes ;  3,  spirilla  of  Finkler  and  Prior ;  4,  spirilla  of  Deneke ;  5,  vibrios  of  Metschni- 
kofF;  6,  spirilla  of  Asiatic  cholera  (Nicolle  and  Morax). 


temperature)  are  favorable,  it  is  half  an  hour ;  in  that  of 
others  it  is  somewhat  longer — from  one  to  two  hours  ;  while 
in  that  of  still  others,  as,  for  instance,  the  tubercle-bacillus, 
it  is  as  much  as  several  days.  When  the  division  always 
takes  place  in  the  same  direction,  and  the  newly  formed  in- 
dividuals remain  attached  to  one  another,  a  chain-like  forma- 
tion results.  In  the  case  of  rods  bacillary  filaments  result,  or 
pseudofilarnents  ;  the  dividing  walls  between  the  individual 
cells  of  the  strand  are  frequently  recognizable  with  difficulty. 
In  the  case  of  spherules  streptococci  {chain-cocci)  result.  (Fig. 
6.)     The  bacteria  may,  however,  after  division  be  arranged, 


MORPHOLOGY  AND   BIOLOGY  OF  BACTERIA.  21 

not  end  to  end,  but  side  by  side  in  groups.     Cocci  thus 

collected    are    designated    staphylococci    (from    araipuXij,   a 

grape),  because  viewed  microscopically  the  cocci  resemble 

bunches  of  grapes.      (Fig.   7,   k.)     When 

the  bacteria  appear  joined  in   pairs,  they        ^     ^^   ^^^ 

are  spoken  of  as   diplococci  (Fig.  7,  U)  or         ^    •..•*    ) 

diplobacilli.     A  number  of  bacteria  divide  /        ^ 

in  two  or  three  successively  vertical  direc-  "^ 

tions.     This   mode  of  division  has    been     Fig- 6.-streptococci. 

observed  only  among  cocci,  and  there  thus 

result  plate-cocci  {tetragenus,  tetrad  arrangement^  and  the 

ball-of-twine-like  packets  known  as  sarcince.     (Fig.  7,  2.) 

In  the  process  of  cell-division  an  essential  developmental 
difference  is  observed  between  bacilli  and  cocci.  The 
bacilli  become  double  their  ordinary  size  before  they  are 
ready  for  multiplication  ;  "while  the  cocci  break  up,  with- 
out preliminary  increase  in  size,  into  two  hemispheres  (or 
in  the  case  of  tetrad  arrangement  into  four  quadrants,  and 
in  the  case  of  sarcina-formation  into  eight  octants),  and 
spheric  cocci  then  form  from  the  products  of  division. 


88 


@^ 


h 


Fig.  7.— Diagram  illustrating  the  morphology  of  cocci:  a.  Coccus  or  micro- 
coqcus ;  b,  diplococcus ;  c,  rf,  streptococci ;  <?,  /,  tetragenococci  or  merismopedia ; 
g,  h,  modes  of  division  of  cocci ;  z,  sarcinae  ;  j,  coccus  with  flagella ;  k,  staphylo- 
cocci (McFarland). 


Vibrios  also  form  pseudofilaments  consisting  of  numerous 
spiral  cells,  and,  besides,  though  seldom,  of  long  spirals 
consisting  each  of  a  single  cell.  Division  occurs  among 
these  in  much  the  same  manner  as  among  bacilli. 

For  every  bacterium  there  is  a  temperature  at  which  it 
thrives  best  (temperature-optiminit),  as  well  as  a  temperature- 
limit  above,  and  beyond  which  it  can  not  suryive  {tempera- 
ture-maximum), and  one  below,  at  which  it  will  just  live 
(temperature -minimum).     Among   pathogenic  bacteria  the 


22  CLINICAL  BACTERIOLOGY. 

temperature-Optimum  is  mostly  about  37°  C.  (98.6°  R). 
There  are  microorganisms,  however,  that  Hve  at  0°  C. 
(32°  F.),  and  exhibit  all  of  their  vital  manifestations,  such 
as  color-formation,  light-development,  peptonization  of 
gelatin,  etc.  Forster  was  the  first  to  obtain  such  bacteria 
in  pure  culture  from  street-dirt,  garden-earth,  sea-water,  and 
from  the  surface  of  luminous  sea-fish.  On  the  other  hand, 
there  are  found,  widely  distributed  in  the  earth,  the  air,  the 
water,  the  contents  of  the  intestine,  etc.,  bacteria  that 
under  aerobic  conditions  require  for  their  growth  tempera- 
tures of  from  50°  C.  (122°  F.)  to  75°  C.  (167°  F.)— so- 
called  thermophilic  bacteria ;  under  anaerobic  conditions, 
however,  these  also  thrive  only  between  34°  C.  (93.2°  F.) 
and  44°  C.  (111.2°  F.). 

The  thermal  death-point  for  individual  mature  bacteria 
at  the  upper  limit,  on  short  exposure,  ranges  for  the  differ- 
ent varieties  between  55°  C.  (131°  R),  60°  C.  (140°  R), 
and  80°  C  (176°  R).     The  death-point  at  the  lower  limit 


CTo^  < o)  O  O  o      C5^3  g=3 

a  b  c  d  e  f 

Fig.  8.— Diagram  illustrating  sporulation :  a,  Bacillus  inclosing  a  small,  oval 
spore  ;  b,  drumstick-bacillus,  with  terminal  spore ;  c,  Clostridium,  with  central  spore ; 
«3f,  free  spores ;  e  and/",  bacilli  escaping  from  spores  (McFarland). 


has  been  determined  only  for  the  smallest  number  of 
growths  ;  many  bacteria  withstand  a  temperature  of  0°  C. 
(32°  F.) — that  is,  freezing — without  injury.  These  state- 
ments are  naturally  not  applicable  to  thermophilic  and 
glacial  microorganisms. 

In  addition  to  dichotomous  division  some  bacteria,  espe- 
cially the  bacilli,  and  only  exceptionally  spirilla,  and  perhaps 
sarcinae,  proliferate  through  the  formation  of  special  perma- 
nent bodies  or  spores.  (Fig.  8.)  In  the  interior  of  the  cell  there 
forms  an  ovoid,  intensely  bright,  and  refractive  body,  the 
spore  J  which  is  enveloped  in  a  firm  capsule,  the  spore-mem- 
brane. One  cell  generally  contains  only  one  spore,  which 
sometimes  is  situated  in  the  middle,  sometimes  at  an  ex- 
tremity. In  the  latter  event,  the  rod  appears  swollen  and 
club-shaped  at  the  corresponding  extremity,  and  it  is  des- 
ignated a  drumstick-bacterium.  (Fig.  8,  ^.)  If  the  swelling 
of  the  bacillus  takes  place  at  the  middle  in  consequence  of 


MORPHOLOGY  AND  BIOLOGY  OF  BACTERIA.     23 

sporulation,  a  spindle-shaped  figure  results,  which  is  known 
as  a  Clostridium. 

At  a  later  stage  the  body  of  the  bacillus  disintegrates, 
and  the  spore  is  set  free.  If  this  finds  its  way  into  a  suita- 
ble nutritive  medium  of  dead  or  living  matter,  it  germinates, 
becomes  a  rod,  the  rod  multiplies  by  dichotomy,  and  the 
whole  process  again  terminates  with  the  formation  of  new 
spores.  The  manner  and  the  method  in  which  the  spore 
becomes  transformed  into  a  rod  vary  among  different  bac- 
teria. Full  details,  in  so  far  as  they  are  of  importance  for 
clinical  bacteriology,  will  be  discussed  in  the  special  section 
of  this  work.  In  general  the  process  of  development  is 
such  that  the  spore  first  takes  up  water,  then  swells,  next 
doubles  in  size,  and  loses  its  marked  refractive  property. 
The  spore-membrane  ruptures  in  the  center  or  at  one  pole  ; 
the  young  bacillus  slips  through  the  rent,  retaining  for 
some  time  the  capsule  of  the  permanent  body. 

Spore-formation  and  spore-germination  take  place  only 
within  certain  temperature-limits,  which  differ  for  each  indi- 
vidual bacterium.  In  the  mode  of  sporulation  just  de- 
scribed the  spore  develops  from  the  interior  of  the  proto- 
plasmic ground-substance  of  the  rod,  and  this  form  of 
fructification  is  therefore  designated  endogeitous  spore -forma- 
tion^ in  contradistinction  from  arthrogenous  spore-formation^ 
where  individual  segments  of  the  cell-chain  assume  spore- 
qualities  without  passing  through  any  intermediate  stage. 
In  external  appearance  these  particular  cells  may  differ  in 
no  respect  from  other  bacteria  of  the  same  chain.  Some- 
times they  become  somewhat  larger  and  brighter  and  more 
highly  refractive  and  surrounded  by  a  firmer  capsule. 

Attempts  have  been  made,  upon  the  basis  of  these  vari- 
ations in  fructification,  to  divide  bacteria  into  two  great 
classes — (i)  those  with  endogenous  spore -formation  {endo- 
sporous  bacterid)  and  (2)  those  with  arthrogenous  spore- 
formation  {arthr OS  porous  bacteria).  Among  the  latter  class 
the  cocci  principally  are  grouped.  However  desirable 
it  might  be  to  classify  the  bacteria  according  to  an  intrinsic 
principle,  it  must  be  emphasized  that  the  classification 
mentioned  appears  at  least  premature,  inasmuch  as  our 
knowledge  concerning  the  arthrogenous  spores  is  as  yet 
but  slight  and  uncertain. 

The  spores  represent  permanent  forms  of  the  bacteria.  . 
Owing  to  the  concentrated  nature  of  their  plasma,  they  are 


24  CLINICAL  BACTERIOLOGY. 

extremely  resistant ;  much  more  so  than  the  mature  bacteria 
themselves.  They  withstand  for  years  drying  and  all  at- 
mospheric influences.  Nature  possesses  but  one  agency 
capable  of  rendering  them  harmless — namely,  the  direct 
action  of  the  rays  of  the  sun,  insolation.  In  order  to  de- 
stroy the  spores  of  anthrax-bacilli,  which  are  not  even 
among  the  most  resistant,  exposure  to  dry  heat  of  140°  C. 
(284°  F.)  for  three  hours,  or  to  steam  of  100°  C.  (212°  F.) 
for  a  few  minutes,  is  necessary.  Far  greater  resistance  is 
displayed  by  the  spores  of  Fliigge's  peptonizing  milk-bac- 
teria, as  well  as  by  some  members  of  the  group  of  hay- 
bacilli  and  potato-bacilli,  which  may  survive  exposure  to 
live  steam  for  five,  six,  and  even  for  sixteen  hours. 

The  question  as  to  the  existence  of  pleomorphic  bacteria 
of  varied  form  has  not  yet  been  decided  with  certainty. 
Pleomorphism  has  been  conceded  by  some  authorities  to 
the  proteus  obtained  by  Hauser  from  decomposing  fluids. 
It  is,  however,  quite  probable  that  the  spheric  bodies  in  the 
cultures  of  the  proteus  result  in  consequence  of  the  retarded 
growth  in  the  presence  of  progressive  division.  If  the  ac- 
tinomyces  are  included  among  bacteria,  there  is  no  alterna- 
tive but  to  admit  the  occurrence  of  pleomorphism.  The 
actinomyces-fungus,  however,  is  no  longer  grouped  by 
most  authorities  among  bacilli.  Kruse,  in  Fliigge's  ''  Text- 
book," makes  a  special  group  of  streptothricece,  of  which 
the  actinomyces  may  be  considered  the  most  important 
representative.  These  streptothriceae  stand  midway  between 
the  filamentous  fungi  and  the  true  bacteria,  and  they  are 
characterized  by  the  formation  ot  filaments  and  especially 
by  the  ramification  resulting  in  consequence  of  their  ger- 
mination. In  old  cultures  the  filaments  break  up  into 
bodies  resembling  bacilli,  spirilla,  and  cocci,  and  there  is 
thus  yielded  an  appearance  of  pleomorphism.  A  further 
peculiarity  of  the  streptothriceae  is  the  formation  of  air- 
hyphae  that  give  rise  to  germ-cells  (spores)  by  segmentation. 
These  are  probably  not  analogous  to  bacterial  spores,  as 
they  are  destroyed  by  exposure  for  five  minutes  to  a  tem- 
perature of  75°  C.  (167°  F.).  Nevertheless,  they  with- 
stand higher  degrees  of  temperature  than  the  filaments^ 
which  undergo  destruction  at  60°  C.  (140°  F.).  As  the 
result  of  personal  investigation,  we  are  of  the  opinion  that 
the  actinomyces-group  represents  a  subdivision  of  the  fila- 
mentous fungi. 


MORPHOLOGY  AND  BIOLOGY  OF  BACTERIA.     25 

Of  late,  attention  has  been  directed  to  ramification,  club- 
formation,  and  granular  disintegration  occurring  among 
tubercle-bacilli  and  diphtheria-bacilli,  and  strongly  sugges- 
tive of  streptothriceae.  Upon  the  basis  of  such  observations 
a  number  of  authorities  have  concluded  that  the  exciting 
agents  of  diphtheria  and  of  tuberculosis,  together  with  the 
whole  actinomyces-group,  belong  to  the  hyphomycetse. 
Such  classification  must,  however,  be  considered  as  prema- 
ture ;  although  the  presumption  is  justified  that  the  tubercle- 
bacillus  and  the  diphtheria-bacillus  stand  in  kindred  rela- 
tionship to  the  ray-fungus  and  the  group  of  organisms 
represented  by  it. 

Involution-changes  must  not  be  confounded  with  variabil- 
ity in  form  of  growth.  The  former  take  place  when  the 
nutritive  medium  is  exhausted  and  the  bacteria  begin  to  die  ; 
the  cells  then  swell,  become  thick  and  plump,  show  defi- 
ciencies, and  undergo  disintegration  and  other  changes  of 
allied  nature.  Whether  all  of  the  resulting  peculiar  forms 
are  to  be  attributed  to  degenerative  changes  is  a  question 
that  must,  at  least,  be  left  open  for  the  present.  It  is  quite 
possible  that  the  very  large,  so-called  giant-cells  result 
rather  in  consequence  of  especially  favorable  vital  conditions. 
Other  involution-manifestations  appear  to  be  due  to  special 
functions  of  the  bacteria,  such  as  their  fermentative  activity. 
In  this  manner  are  perhaps  to  be  explained  the  involution- 
forms  of  the  acetic  bacterium,  and  especially  of  numerous 
microbes  that  are  cultivated  in  media  containing  grape-sugar. 

Bacteria  are  ubiquitous :  they  are  found  everywhere  ; 
only  the  internal  organs  of  the  human  and  the  animal  body 
not  in  communication  with  the  atmosphere  are  free  from 
them.  Some  of  the  pathogenic  germs  confine  their  activi- 
ties to  certain  living  organisms,  so  that  their  area  of  dis- 
tribution is,  in  consequence,  a  restricted  one. 

For  their  sustenance  the  bacteria  require  preformed  or- 
ganic carbon-compounds,  as  most  of  them,  by  reason  of 
their  deficiency  in  chlorophyl,  are  capable  of  utilizing  the 
carbon  dioxid  of  the  atmosphere.  They  require  also  nitro- 
gen-compounds, which  they  can  obtain  from  organic  sub- 
stances, as  well  as  from  inorganic  nitrates  and  ammonia- 
compounds.  It  need  scarcely  be  added  that  water  is  neces- 
sary for  the  development  of  the  bacteria.  The  nutritive 
material  for  the  bacteria  must  be  feebly  alkaline  or  neutral, 
as  in  general  they  do  not  develop  so  well  upon  acid  media. 


26  CLINICAL  BACTERIOLOGY. 

Light  is  required  only  by  such  species  of  bacteria  as 
contain  chlorophyl.  For  the  far  greater  number,  the 
action  of  sunhght  and  even  of  diffuse  dayhght  is  directly 
injurious  ;  their  growth  is  inhibited,  and  after  a  time  the 
bacteria  are  entirely  destroyed  by  the  action  of  the  light. 
This  sterihzing  influence  of  light  is  most  manifest  when  the 
rays  of  the  sun  fall  vertically  upon  the  surface  of  the  cul- 
tures. Not  only  the  growing  forms,  but  also  the  perma- 
nent forms,  the  spores,  succumb  to  this  action  of  light. 
The  spores  of  anthrax,  for  instance,  are  destroyed  in  a 
moist  substratum  by  the  action  of  light  in  a  somewhat 
shorter  time  than  the  mature  bacilli.  In  the  dried  state, 
however,  the  spores  prove  more  resistant  to  the  rays  of  the 
sun.  The  metabolic  products  of  the  bacteria  also  become 
materially  attenuated  under  the  influence  of  strong  light, 
especially  when  there  is  no  obstacle  to  access  of  oxygen. 
Nutritive  media  that  have  been  exposed  to  light  are  not,  in 
consequence,  unsuitable  for  the  cultivation  of  microorgan- 
isms. Not  all  rays  of  the  spectrum  are  bactericidal,  but 
only  the  blue,  the  violet,  and  the  ultraviolet. 

Of  great  importance  is  the  influence  of  oxygen.  Many 
bacteria  are  capable  of  developing  only  in  the  presence  of 
this  gas  (aerobic  bacterici) ;  others,  on  the  other  hand, 
thrive  only  in  complete  absence  of  oxygen  {anaerobic  bac- 
teria)) ;  a  third  group  occupies  an  intermediate  position  : 
they  grow  as  well  in  the  presence  as  in  the  absence  of  oxy- 
gen (facultative  anaerobic  bacterid). 

In  their  growth  and  multiplication  the  bacteria  generate 
metabolic  products  upon  and  from  their  culture-medium. 
Thus  the  whole  range  of  fermentative  processes  is  an  ex- 
pression of  bacterial  activity,  and  the  substances  that  are 
thereby  formed  are  to  be  viewed  as  the  metabolic  products 
of  fermentative  bacteria.  The  same  statement  applies  to 
putrefaction  and  decomposition. 

A  number  of  mostly  innocuous  bacteria  are  characterized 
by  the  formation  of  pigment.  These  pigment-bacteria  pro- 
duce most  varied  coloring -matters,  which  appear  in  all  pos- 
sible shades  of  color  only  when  access  of  air  is  entirely 
unobstructed.  Some  bacteria  give  rise  in  their  culture- 
media  to  beautiful  fluorescence,  others  to  phosphorescence — 
that  is,  they  appear  luminous  in  the  dark. 

The  chemic  combinations  to  which  bacteria  give  rise  in 
artificial  nutritive  media  are  at  present  of  preeminent  in- 


MORPHOLOGY  AND  BIOLOGY  OF  BACTERIA.     27 

terest.  The  ability  of  various  bacteria  to  effect  chemic 
decomposition  may,  from  the  present  state  of  knowledge, 
be  considered  as  extremely  varied.  The  most  profound  re- 
ductions, and  the  most  far-reaching  oxidations,  the  forma- 
tion of  most  complex  combinations  and  their  disintegration 
into  atoms,  are  effected  by  bacteria.  From  nitrates  are 
formed  nitrites,  free  nitrogen,  and  ammonia ;  on  the  other 
hand,  bacteria  in  the  earth  are  capable  of  forming  nitrates 
from  nitrogen  and  ammonia.  The  bacteria  of  earth  which 
are  responsible  for  the  process  last  named — the  so-called 
nitrification-process — are  of  great  biologic  interest.  They 
oxidize  the  ammonia — the  final  product  of  the  de- 
composition of  nitrogenous  substances — into  nitrites,  and 
these  further  into  nitrates,  which  then  enter  into  the  forma- 
tion of  new  plants.  Winogradsky  recognized  two  varieties 
— the  7iitrosobacteria  (niirosococcus  and  nitrosomonas), 
which  oxidize  ammonia  into  nitrites,  but  not  further ;  and  the 
nitrobacteria,  which  are  incapable  of  acting  upon  the  am- 
monia, but,  on  the  other  hand,  convert  the  nitrites  into 
nitrates.  Both  species  are  characterized  by  the  remarkable 
fact  that  they  grow  only  in  culture-media  that  contain  no 
trace  of  organic  carbon-compounds — that  is,  they  are 
capable  of  obtaining  the  carbon  they  require  from  the  car- 
bon dioxid  of  the  atmosphere,  without  the  aid  of  chloro- 

Under  appropriate  nutritive  conditions  bacteria  de- 
velop hydrogen  sulphid  from  all  sulphur-containing  com- 
pounds. The  so-called  sulphur-bacteria  (beggiatoa,  thio- 
thrix)  require  a  supply  of  hydrogen  sulphid,  extracting  the 
sulphur  and  storing  it  up  in  their  protoplasm  in  the  form  of 
bright  granules.  With  deficiency  of  nutritive  material  the 
sulphur-granules  are  oxidized  into  sulphuric  acid. 

Urea  is  decomposed  into  carbon  dioxid  and  ammonia. 
There  is  scarcely  an  organic  combination  from  which  these 
gases  and  also  free  hydrogen  may  not  finally  be  produced 
through  bacterial  activity. 

Of  especial  significance  is  the  bacterial  decomposition  of 
substances  that  serve  for  the  nutrition  and  for  the  construc- 
tion of  the  body.  Starch  is  dextrinized,  saccharified,  even 
subjected  to  mucoid  transformation.  Sugar  undergoes  vari- 
ous forms  of  fermentation  in  consequence  of  bacterial  activ- 
ity ;  alcohol,  carbon  dioxid,  acetic  acid,  lactic  acid,  butyric 
acid,  succinic  acid,  and    others,   have  been  demonstrated 


28  CLINICAL   BACTERIOLOGY. 

among  the  products  of  fermentation.  Not  only  grape-sugar, 
but  also  numerous  other  varieties  of  sugar  (lactose,  galactose, 
arabinose,  etc.)  may  undergo  fermentation  in  consequence  of 
the  activity  of  certain  kinds  of  bacteria.  Fats  are  split  up 
into  fatty  acids  and  glycerin.  Much  study  has  been  devoted 
to  the  changes  that  albuminoid  substances  undergo  as  a  re- 
sult of  the  activity  of  bacteria,  and  that,  accordingly  as 
more  or  less  fetid  gases  are  generated,  are  designated 
putrefaction  or  decomposition.  From  the  aromatic  group 
of  the  albumin-molecule  there  result  aromatic  amidoacids 
(tyrosin,  amidophenylpropionic  acid,  etc.)  and  certain  ben- 
zol-derivatives, which  impart  to  some  bacterial  cultures  and 
to  the  feces  their  characteristic  offensive  odor  (phenol,  indol, 
skatol,  etc.). 

From  the  group  of  fatty  bodies  of  albumin  the  bacteria 
form,  in  addition  to  fatty  acids,  oxyacids,  and  amidofatty 
acids  (leucin,  glycocol,  alanin),  a  large  number  of  organic 
toxic  bases,  which  are  designated  ptomains,  and  for  our 
knowledge  of  which  we  are  indebted  to  the  investigations 
of  Selmi,  Gautier,  Nencki,  and  Brieger.  The  ptomains  be- 
long to  the  group  of  amins  and  ammonium-bases — for 
instance,  cadaverin  (pentamethylendiamin),  putrescin  (tetra- 
methylendiamin),  cholin,  betain,  neurin,  and  muscarin  ;  of  a 
portion  of  these  substances  only  the  chemic  formula  has 
been  determined,  although  their  constitution  is  as  yet  un- 
known— for  example,  saprin,  gadinin,  etc. 

A  knowledge  of  the  metabolic  products  named  is  of  the 
highest  diagnostic  significance  in  bacteriologic  investigation. 
It  is  important  to  know  whether  a  bacterium  gives  rise 
to  the  formation  of  acid  or  alkali,  whether  it  causes  coagu- 
lation of  milk  and  liquefies  gelatin  by  peptonization,  whether 
it  generates  phenol  and  indol,  to  what  extent  it  reduces 
nitrates,  whether  it  forms  gases,  an-d  which.  All  of  these 
peculiarities  may  be  of  determining  significance  for  the  iden- 
tification of  a  given  bacterium. 

Of  considerable  importance  are  the  chemic  activities  dis- 
played by  bacteria  in  the  domain  o(  physiology  ^vid  oi  pathol- 
ogy. Bacteria  participate  in  the  normal  course  of  the  diges- 
tive process  in  the  hum^n  intestine  ;  the  interesting  experi- 
ment of  Thierfelder  and  Nuttall,  who,  with  complete  exclu- 
sion of  all  bacteria,  fed  with  sterile  nourishment  a  young 
animal  removed  sterile  by  Cesarean  section  from  the  uterus 
of  a  pregnant  mother,  demonstrates  that  this  participation 


MORPHOLOGY  AND   BIOLOGY  OF  BACTERIA.  29 

of  bacteria  in  the  process  of  digestion  is  not  indispensable. 
On  the  other  hand,  the  far-reaching  bacterial  decomposi- 
tion of  albumin,  the  formation  of  organic  acids  and  of  an 
abundance  of  gaseous  products,  not  rarely  give  rise  to 
severe  digestive  disturbances.  The  formation  of  hydrogen 
sulphid  may  cause  intoxication  from  the  gastro-intestinal 
tract  (hydrothionemia),  and  in  individual  cases  also  hydro- 
thionuria.  Bacterial  decomposition  of  urea  is  the  cause  of 
cystitis  ;  and  the  formation  of  gas  in  the  bladder  by  bacteria 
may  result  in  pneumaturia.  Cadaverin  and  putrescin  may 
result  from  putrefaction  in  the  intestine  or  in  bronchiectatic 
cavities  ;  when  certain  intestinal  mycoses  exist,  these  sub- 
stances appear  in  the  urine   (cystinuria). 

Of  far  greater  and  more  comprehensive  importance,  how- 
ever, are  those  metabolic  products  that  are  produced  in  the 
tissues  of  the  body  by  the  exciting  agents  of  disease,  the 
specific  toxins,  which  give  rise  to  the  toxic  constitutional 
manifestations  of  the  infectious  diseases.  These  poisons  are 
generated  also  by  individual  bacteria  in  artificial  cultures. 
Loffler  pointed  out  that  by  means  of  glycerin  a  chemic 
poison  precipitable  by  alcohol  could  be  extracted  from  cul- 
tures of  diphtheria-bacilli,  and  with  which  the  disease  could 
be  induced  in  animals  in  a  manner  analogous  to  that  in 
which  it  is  effected  by  the  bacilli  themselves.  Roux  and 
Yersin  demonstrated  that  this  diphtheria-poison  is  destroyed 
by  a  temperature  of  ioo°  €.(212°  F,).  They  evaporated  the 
poison-containing  bouillon  at  low  temperatures,  and  ob- 
tained a  residue  that  was  readily  soluble  in  water,  and 
highly  toxic.  The  alcoholic  extract  proved  innocuous. 
The  poison  is  thus  insoluble  in  alcohol,  and  is  precipitable 
by  this  out  of  watery  solution.  On  dialysis  it  passed  very 
slowly  through  animal  membrane.  Roux  and  Yersin 
demonstrated  further  that  the  toxin  of  the  filtered  bouillon 
was  most  completely  carried  down  with  a  calcium-chlorid 
precipitate.  Attached  to  this  precipitate  and  dried  with  it, 
the  toxin  proved  much  more  resistant  to  heat.  The  poison 
proved  active  only  when  used  subcutaneously  or  intra- 
venously, while  when  administered  by  the  mouth,  it  was 
harmless.  Its  action  was  specific,  the  palsies  characteristic 
of  diphtheria  taking  place.  From  the  evidence,  the  French 
investigators  considered  the  poison  a  ferment. 

In  Germany,  Brieger  and  C.  Frankel  later  studied  care- 
fully the  diphtheria-toxin,  as  well  as  the  toxins  of  numerous 


30  CLINICAL  BACTERIOLOGY. 

other  bacteria,  and  especially  the  tetanus-toxin.  They  pre- 
pared the  toxic  substances  either  by  evaporation  of  filtered 
bouillon-cultures  in  a  vacuum  at  temperatures  of  from  20° 
C.  (68°  F.)  to  35°  C  (95°  R),  and  by  precipitation  of  the 
concentrated  bouillon  with  absolute  alcohol ;  or  they  super- 
saturated the  bouillon-filtrate  with  ammonium  sulphate  or 
sodium  phosphate,  and  obtained  from  the  precipitate  the 
poison,  which  proved  to  be  not  dialyzable.  The  poisons 
thus  obtained  by  alcoholic  precipitation  or  separation  by 
means  of  salts  yielded  albuminoid  reactions.  They  could 
not  be  included  among  the  globulins,  as  they  were  precipi- 
table  out  of  the  bouillon-filtrate  by  the  two  salts  named 
only,  and  not  by  magnesium  sulphate.  Brieger  and  C. 
Frankel  named  the  toxic  substances  obtained,  and  which 
appeared  as  amorphous  powders,  toxalbumins.  These  are 
destroyed  by  temperatures  of  60°  C.  (140°  F.) ;  according 
to  Buchner,  the  presence  of  neutral  salts  renders  them 
somewhat  more  resistant.  These  albuminoid  powders  cer- 
tainly contain  the  specific  bacterial  poison  ;  with  a  minimal 
amount  of  the  powder  obtained  in  this  manner  from  tetanus- 
bouillon,  for  instance,  one  can  induce  typical  tetanus  in 
animals.  Nevertheless,  the  bacterial  poisons  can  not  with 
certainty  be  considered  as  albuminoid  bodies.  It  is  rather 
more  probable  that  the  actual  active  bacterial  poisons 
are  only  carried  down  in  the  preparation  of  these  albu- 
minoid powders,  and  that  they  adhere  to  the  albumin  in  a 
purely  mechanical  manner,  as  it  was  possible  to  obtain  with- 
out difficulty  specifically  acting  toxins  by  the  growth  of 
pathogenic  bacteria  in  nutritive  media  free  from  albumin. 
(See  Uschinsky's  Nutritive  Medium,  p.  87.) 

In  the  course  of  further  investigations  of  the  tetanus- 
toxin  Brieger  and  Cohn  also  soon  came  to  the  conclusion 
that  this  substance  represents  no  true  albuminoid  body.  A 
notable  advance  in  the  knowledge  of  the  nature  of  bacterial 
toxins  was  made  through  the  investigations  of  Brieger  and 
Boer.  These  investigators  elicited  the  fact  that  the 
poisons  of  diphtheria  and  of  tetanus  are  precipitated  from 
their  solutions,  from  filtered  bouillon-cultures,  by  means  of 
heavy  metals,  in  the  form  of  more  or  less  soluble  double 
combinations.  A  one  per  cent,  solution  of  zinc  chlorid 
proved  most  available,  being  added  in  double  the  amount 
of  the  toxin-solution.  The  double  zinc-combinations  thus 
resulting  no  longer  exhibit  any  of  the  well-known  albu- 


MORPHOLOGY  AND   BIOLOGY  OF  BACTERIA.  31 

minoid  reactions.  They  are,  however,  in  no  less  degree 
toxic,  so  that  there  can  be  no  doubt  that  they  contain  the 
true  specific  poison.  They  are  insoluble  in  distilled  water, 
but  readily  soluble  in  feebly  alkaline  water  or  water  con- 
taining sodium  chlorid  ;  they  are  most  readily  destroyed 
by  acids,  but,  on  the  other  hand,  they  are  unaffected  by 
substances  of  a  feebly  alkaline  reaction.  For  the  details 
of  the  preparation  and  of  the  properties  of  the  toxins  of 
diphtheria  and  of  tetanus  reference  may  be  made  to  the 
proper  chapters  in  the  special  section. 

To  be  separated  from  these  toxins,  formerly  designated 
toxalbumins,  and  which  have,  up  to  the  present,  been  ob- 
tained in  a  manner  perfectly  free  from  criticism  only  in  the 
two  toxic-infectious  diseases,  diphtheria  and  tetanus,  and 
recently  in  botulism,  are  those  poisons  that  are  contained 
within  the  bodies  of  the  bacteria  themselves.  R.  Pfeiffer 
first  obtained  these  from  cholera-vibrios  and  typhoid- 
bacilli,  by  destroying  fresh  agar  smear-cultures  through 
the  action  of  chloroform-vapor,  or  through  exposure  to  a 
temperature  of  54°  C.  (129.2°  F.)  for  an  hour.  These 
poisons,  in  contrast  with  the  virus  of  diphtheria  and  of  teta- 
nus, are  not  demonstrable  in  the  filtered  cultures.  They  are, 
however,  like  the  former,  unstable  in  nature.  Exposure 
to  temperatures  above  60°  C.  (140°  F.)  markedly  reduces, 
without,  however,  completely  abolishing,  their  activity. 
According  to  PfeifTer's  view,  there  remain  secondar}^  toxins 
that  prove  to  be  much  more  stable,  withstanding  boiling 
for  several  hours,  but  which  are  from  ten  to  twenty  times 
less  toxic.  A  still  further  important  difference  exists 
between  these  intracellular  poisons  of  typhoid  fever  and  of 
cholera  and  the  toxins  of  the  pure  intoxication -diseases — 
diphtheria  and  tetanus  ;  while  the  former  manifest  their 
activity  in  animals  immediately  after  their  introduction  and  at 
once  induce  disease-symptoms,  the  latter  only  give  rise  to 
their  toxic  manifestations  after  a  well-defined  period  of  in- 
cubation. 

The  physiologic  properties  of  the  poisons  of  typhoid  fever 
and  of  cholera  will  be  thoroughly  considered  in  the  special 
section. 

Finally,  those  toxic  substances  must  yet  be  mentioned 
that  are  known  as  bacterial  proteins  (Buchner).  These  differ 
from  the  poisons  already  mentioned  in  withstanding  a  boil- 
ing temperature,   and   they  appear,   in   contrast  with   the 


32  CLINICAL  BACTERIOLOGY. 

specific  bacterial  poisons,  to  be  the  same  in  many  or  in  all 
bacteria  ;  at  least,  their  action  is  not  specific.  They  never 
give  rise  to  the  typical  clinical  picture  that  is  characteristic 
of  the  bacteria  from  which  they  are  obtained,  but  they 
always  give  rise  to  fever  and  leukocytosis  only,  and,  on 
subcutaneous  injection,  to  local  inflammation  and  suppura- 
tion besides.  For  the  white  blood-corpuscles  they  possess 
especially  a  most  marked  attractive  influence,  being  with 
regard  to  them  positively  chemotactic.  Administered  in 
large  amounts  the  bacterial  proteids  cause  the  death  of  the 
animal ;  but  even  then  the  course  of  the  disease  presents 
nothing  characteristic,  and  not  more  so  the  postmortem 
findings.  Detailed  reference  to  the  latter  will  be  made  in 
the  consideration  of  tuberculin  (see  Tuberculosis),  which, 
like  mallein  (see  Glanders),  is  a  proteid. 

Chemically,  these  bacterial  proteids  approach  the  vege- 
table caseins.  Treated  with  basic  aniline  dyes,  they  lose 
their  toxic  activity,  and  they  may  be  considered  as  that 
constituent  of  the  bacterial  cell  that  confers  upon  it  the 
property  of  taking  up  stains.  The  bacterial  proteids  are 
soluble  in  dilute  alkalies,  and  insoluble,  on  the  other  hand, 
in  dilute  acids.  They  are  best  prepared  by  adding  to  a 
bouillon-culture  in  as  large  amount  as  possible  bacterial 
scrapings  from  solid  culture-media,  and  boiling  the  mixture 
for  about  two  hours,  then  evaporating  down  to  one-fifth  or 
one-fourth,  and  filtering  through  porcelain.  From  the 
filtrate  the  proteid  substances  are  precipitated  by  the  addi- 
tion of  ten  times  their  volume  of  absolute  alcohol.  They 
form  an  amorphous  powder  that  is  readily  soluble  in  water. 
Besides,  the  filtrate  of  unboiled  old  bouillon-cultures  gener- 
ally contains  a  certain  amount  of  proteid  substances  that 
have  gradually  found  their  wa)^  out  of  the  bodies  of  the 
numerous  dead  bacteria  into  the  fluid. 

E.  Buchner  has  recently  devised  a  procedure  for  the 
preparation  of  unchanged  cell-fluid.  The  cells  are  rubbed 
up  mechanically  and  exposed  to  a  pressure  of  from  four 
to  five  hundred  atmospheres.  Buchner  obtained  in  this 
way  from  yeast-cells  a  fluid  of  yellow  color  and  alkaline 
reaction  that  yielded  ten  per  cent,  of  solid  constituents, 
and  an  abundance  of  albuminous  bodies  precipitable  by 
heat.  This  so-called  yeast-cell  expressed  fluid  no  longer 
contained  living  germs,  although  it  still  was  capable  of  in- 
ducing alcoholic  fermentation.     H.  Buchner  considers  the 


INFECTION.  33 

substance  present  in  the  expressed  cell-fluid,  which  pos- 
sesses the  properties  of  fermenting  sugar,  as  parablastic, 
and  designates  it  zymase.  This  zymase  is  produced  within 
the  cell,  and  H.  Buchner  compares  the  entire  process  with 
the  production  and  the  action  of  the  toxins  of  tetanus  and 
diphtheria,  which  likewise  are  formed  within  the  bacterial 
cell. 

Some  species  of  bacteria  are  capable  of  thriving  not  only 
upon  dead,  but  also  upon  living,  matter,  in  man  as  well  as 
in  animals.  Gaining  entrance  into  a  living  organism,  these 
bacteria  multiply  at  the  expense  of  their  host,  and,  with 
the  aid  of  their  metabolic  products,  unfold  their  deleterious 
activity  ;  the  affected  individual  is  made  ill.  Bacteria  capa- 
ble of  inducing  disease  are  designated  pathogenic ;  those, 
on  the  other  hand,  that  are  innocuous  and  harmless  are 
designated  nonpathogenic.  Those  that  are  capable  of  under- 
going multiplication  only  within  a  higher  living  organism 
are  known  as  parasites  (true,  strict,  obligate  parasites).  In 
contrast  with  these  are  saprophytes,  those  bacteria  that 
thrive  only  upon  dead  material.  There  is,  however,  no 
sharp  division  between  parasites  and  saprophytes.  Many 
bacteria  are  adapted  to  both  modes  of  life  :  these  are  facul- 
tative parasites — that  is,  they  live  only  temporarily  within 
the  animal  body,  but  usually  external  to  it,  in  the  earth  or 
in  water.  On  the  other  hand,  many  of  the  especially  patho- 
genic bacteria  are  essentially  parasitic.  By  means  of  our 
culture-media  we  have,  however,  succeeded  in  cultivating 
them  outside  the  body,  and  have  thus,  by  artificial  means, 
made  them  facultative  saprophytes. 


n*  INFECTION. 

Those  diseases  are  designated  infectious  that  result 
through  the  vital  activity  of  vegetable  or  animal  micro- 
organisms. The  causative  agents  of  the  majority  of  the 
diseases  included  in  this  group  belong  to  the  class  of  bac- 
teria. The  filamentous  fungi,  as  well  as  the  lower  forms 
of  animal  life  (protozoa),  have  until  now  played  a  less 
important  role  in  the  etiology  of  disease.  Formerly,  the 
infectious  diseases  were  subdivided  into  contagious  and 
miasmatic.  With  the  former,  transmission  takes  place 
MkixoM^  contagion — that  is,  through  the  conveyance  of  the 
3 


34       •  CLINICAL  BACTERIOLOGY. 

materies  morbi  from  the  sick  to  the  well.  With  the  latter, 
the  disease-virus,  the  miasm,  is  taken  up  only  from  the  air, 
or,  in  general,  from  surrounding  nature  ;  the  miasmatic  dis- 
eases are  never  transmitted  from  individual  to  individual. 
This  division  has  now  lost  much  of  its  significance.  Most 
infectious  diseases  with  whose  causative  agents  we  are 
familiar  are  contagious-miasmatic — that  is,  they  are  trans- 
mitted as  well  from  one  person  to  another  as  from  without, 
through  the  intermediation  of  the  air,  the  water,  etc.  Even 
malaria  may  no  longer  be  considered  a  strictly  miasmatic 
infection,  although  under  natural  conditions  it  is  probably 
never  communicated  from  one  person  to  another,  since  Ger- 
hardt  has  transmitted  the  disease  from  a  malarial  patient  to 
a  healthy  person  by  blood-transfusion. 

According  to  the  activity  of  their  causative  bacteria,  in- 
fections may  be  divided  into  toxic  and  infectious.'^  In  the 
former  the  manifestations  caused  directly  by  the  living 
germ — that  is,  the  local  symptoms  at  the  site  of  infection — 
are  subordinate  to  the  toxic  manifestations — those  result- 
ing from  absorption  of  the  poisonous  substances  generated 
by  the  bacteria.  As  examples  of  such  toxic  infections  may 
be  mentioned  experimental  diphtheria  in  animals,  and  in 
man  especially  tetanus,  the  local  manifestation  of  which 
often  consists  only  in  slight  suppuration,  or  is  entirely  ab- 
sent, so  that  the  site  of  infection  frequently  escapes  detec- 
tion. On  the  other  hand,  in  the  infectious  diseases  the 
disease -germs  themselves  play  the  most  important  role, 
acting  especially  through  their  enormous  multiplication. 
If  this  takes  place  throughout  the  entire  body  by  way  of  the 
blood-stream,  the  condition  is  designated  septicemia.  The 
type  of  such  a  condition  is  anthrax,  in  which,  no  matter  in 
what  situation  the  infection  took  place,  the  bacillus  may  be 
found  present  everywhere — in  every  organ  and  in  every 
tissue. 

Examples'  of  infectious  diseases  in  a  restricted  sense  are, 
in  man,  for  instance,  cholera  and  pneumonia,  in  which  enor- 
mous multiplication  of  the  causative  agents  takes  place 
within  a  circumscribed  area  (intestine  or  lung),  and  the 
local  symptoms  are  very  considerable.     Just  these  two  in- 

*  This  nomenclature  is  awkward  and  tautologic.  The  differences  intended 
to  be  expressed  are  of  degree  and  not  of  kind.  All  of  the  diseases  of  this 
group  are  infectious,  naturally  in  varying  grade,  and  all  give  rise  to  secondary 
intoxication,  also  of  varying  grade. — A.  A.  E. 


INFECTION.  35 

stances,  however,  teach  that  the  distinction  between  toxic 
and  infectious  diseases  is  not  absolute,  but  only  relative. 
Pneumonia  is  not  unattended  with  constitutional  symptoms 
(fever,  albuminuria,  etc.)  due  to  the  absorption  of  the  bac- 
terial toxins  circulating  in  the  blood ;  and,  also,  the  pro- 
found symptoms  of  the  algid  stage  of  cholera  are  only 
explicable  on  the  assumption  of  a  poison  generated  by  the 
vibrios  in  the  intestine,  and  thence  absorbed  into  the  circu- 
lation. Even  in  the  true  septicemias  the  disease  and  death 
are  ultimately  not  alone  caused  mechanically  by  the  indefi- 
nite multiplication  of  the  bacteria,  but  here,  also,  the  chemic 
activity  of  the  bacteria — their  production  of  poisons — 
comes  into  play.  Upon  the  other  hand,  even  in  the  case 
of  tetanus,  the  most  perfect  representative  of  the  toxic  in- 
fections, it  has  been  demonstrated  that  the  influence  of 
bacteriarmultiplication  in  the  course  of  natural  infection  is 
not  entirely  wanting.  The  multiplication  of  the  germs, 
however,  is  quite  insignificant  and  transitory,  and  the  re- 
markable activity  of  the  poison  dominates  the  clinical 
picture. 

The  mechanical  factor  plays  a  much  more  important  role 
in  so-called  pyemia  than  in  the  case  of  septicemia.  In 
the  former  condition  the  bacteria — generally  pyogenic 
microorganisms  or  those  inducing  inflammation — likewise 
gain  entrance  into  the  blood,  by  way  of  the  lymphatic  chan- 
nels, from  a  local,  primary  focus  of  disease.  They  do  not, 
however,  become  generalized,  but  remain  within  certain 
organs,  at  times  in  the  serous  membranes,  at  other  times  in 
the  joints,  at  yet  other  times  in  the  skin,  in  the  liver,  the 
spleen,  the  kidneys,  the  myocardium,  etc.  The  bacterial 
masses  circulating  in  the  blood  cause  occlusion  in  larger  or 
smaller  arterial  areas,  and  there  result  in  this  way  infarcts, 
ischemic  softening,  abscesses.  Pyemia  is  thus  to  be  consid- 
ered essentially  a  consequence  of  metastases  of  the  causative 
agents  of  the  disease.  In  each  of  the  metastases,  however, 
the  bacteria  again  give  rise  to  their  toxic  products  ;  which, 
in  turn,  contribute  to  the  further  course  of  the  disease. 

Perhaps  many  of  the  diseases  caused  by  filamentous  fungi 
(mycoses)  depend  upon  purely  mechanical  lesions,  with- 
out constitutional  intoxication  of  the  organism  ;  but  in 
those  diseases  caused  by  bacteria  the  influence  of  poison- 
production  is  of  importance  in  every  case  :  every  infection 
is  attended  also  with  intoxication ;  only  the  more  marked 


36  CLINICAL   BACTERIOLOGY. 

prominence  or  recession  of  the  latter  justifies  the  division 
into  infectious  and  toxic  infections. 

The  term  infection  indicates,  in  verbal  expression,  the 
entrance  of  microorganisms  into  the  animal  body.  With 
the  mere  entrance  of  the  disease-germs  into  the  body  the 
infection — that  is,  the  generation  of  the  disease — is,  how- 
ever, by  no  means  completed.  The  bacteria  taken  up  may 
again  leave  the  body  :  may,  for  instance,  pass  through  the 
entire  digestive  tract  without  causing  the  slightest  injury. 
Thus,  cholera-bacilli  may  temporarily  be  found  in  the  in- 
testinal evacuations  of  healthy  individuals,  and  tetanus- 
bacilli  may  be  demonstrable  in  the  intestinal  contents  of 
healthy  animals.  The  germs  taken  up  may,  however, 
remain  latent  at  the  same  place  at  which  they  later  induce 
disease,  without  doing  this  in  the  first  instance.  Thus, 
bacteria,  and  especially  those  that  we  shall  further  on  learn 
to  be  the  cause  of  inflammation  and  suppuration,  are  present 
normally  not  only  upon  the  entire  cutaneous  surface,  but 
also  in  the  mouth,  in  the  upper  air-passages,  in  the  entire 
digestive  tract,  in  the  lower  portion  of  the  genito-urinary 
apparatus,  in  the  external  ear,  in  the  eye — in  short,  where- 
ever  the  external  air  has  unobstructed  access.  The  unin- 
jured skin,  however,  as  well  as  the  normal  mucous  mem- 
brane, does  not  permit  the  bacteria  to  force  their  way  beneath 
the  surface ;  and  if  they  should  multiply  upon  the  surface 
and  generate  poisons — as,  for  instance,  putrefactive  bacteria 
certainly  do  in  the  intestine — these  poisons,  in  the  presence 
of  a  normal  mucous  membrane,  are  either  not  at  all  ab- 
sorbed or  not  as  such.  A  lesion  of  the  skin  or  of  the  mucous 
membrane  is  first  necessary  to  permit  the  entrance  of  the  bac- 
teria into  the  actual  interior  of  the  body  (which  normally  is 
free  from  germs)  and,  thereby,  the  development  of  infection. 
Nor  is  the  entrance  of  bacteria  into  the  interior  of  the  body 
by  any  means  synonymous  with  infection.  Even  if  a  lesion 
has  been  induced,  and  if  bacteria  have  gained  entrance  into 
the  tissues  of  the  body,  the  disease  may  not  be  developed. 
The  bacteria  may  be  taken  up  by  cells  of  the  body  and  be 
destroyed  (phagocytosis) ;  or  they  may  succumb  to  the 
bactericidal  property  often  possessed  by  the  blood  and  the 
fluids  of  the  tissues.  They  may,  further,  remain  free  and, 
in  a  certain  degree,  inactive  until  they  die  ;  or,  possibly, 
induce  the  disease  at  a  later  period.  Thus,  as  has  been 
demonstrated    by  an    accidental    observation   of  Vaillard, 


INFECTION.  37 

infectious  tetanus-material  may  gain  entrance  into  a  mem- 
ber, there  remain  without  effect,  and  when,  later,  a  con- 
tusion or  some  other  injury  of  the  member  takes  place, 
tetanus  may  long  afterward  occur.  A  similar  latent  period  is 
probably  also  responsible  for  the  observation  recently  made 
of  the  presence  of  tubercle-bacilli  in  the  lymph-glands  of 
apparently  healthy  individuals.  Thus,  the  entrance  of  bac- 
teria into  the  body  does  not  invariably  give  rise  to  infec- 
tion ;  but,  on  the  contrary,  the  conjunction  of  a  whole  series 
of  circumstances  is  necessary  in  order  that  infection  may 
take  place.  Of  such  circumstances,  which  are  related  partly 
to  the  infecting  material  and  partly  to  the  infected  indi- 
vidual, the  following  are  thus  far  known  : 

(a)  The  Virulence  of  the  Infectious  Agent. — The  viru- 
lence of  bacteria  is  a  varying  one.  The  degree  of  toxicity 
possessed  by  a  bacterial  culture  obtained  from  the  diseased 
focus  decreases  progressively  :  in  the  case  of  some  bacteria 
more  rapidly  (as,  for  instance,  diphtheria-bacilli,  and  most 
rapidly  pneumococci)  ;  in  the  case  of  others,  more  slowly 
(an  anthrax-culture,  for  instance,  will  remain  toxic  for 
weeks,  a  tetanus-culture  for  many  months  ;  a  culture  of 
tubercle-bacilli,  with  suitable  transplantation,  will  still  be 
capable  of  infection  after  the  lapse  of  years).  The  reduc- 
tion in  virulence  is  frequently  accompanied  by  a  corre- 
sponding reduction  in  activity  of  growth,  though  not 
always.  Diphtheria-bacilli,  for  instance,  are  said  to  grow 
more  luxuriantly  upon  artificial  culture-media  the  more 
their  virulence  is  diminished.  The  variation  in  degree  of 
the  virulence  of  bacteria  can,  therefore,  not  be  dependent 
upon  their  varying  activity  of  growth.  The  virulence  must 
rather  be  considered  to  correspond  with  the  capability  of 
toxin-production  :  the  more  virulent  bacterium  generates  a 
more  active  poison  or  a  greater  amount  thereof  than  the 
less  virulent  organism.  The  reduction  in  virulence  in  arti- 
ficial culture  is  prevented  in  the  case  of  many  bacteria  by 
frequent  transplantation  upon  fresh  nutritive  media  ;  it  may, 
therefore,  be  in  some  way  related  to  exhaustion  of  the 
culture-medium,  a  want  of  appropriate  nutritive  material, 
an  accumulation  of  inhibiting  metabolic  products.  Such 
an  interpretation  is  applicable  also  to  the  marked  reduction 
in  virulence  that  is  often  observed  in  culture-media  con- 
taining grape-sugar  ;  the  process  of  fermentation  appearing 
to  influence  the  media  in  a  manner  unfavorable  for  toxin- 


38  CLINICAL  BACTERIOLOGY. 

production.  Artificial  means  for  reducing  the  virulence  of 
a  culture  include  heat  (p.  40),  light,  electricity,  as  well  as 
various  chemic  influences — for  instance,  iodin  trichlorid  with 
diphtheria  and  tetanus. 

When  the  virulence  of  a  bacterial  culture  has  once  been 
attenuated,  it  may  again  be  intensified  by  passage  of  the 
bacteria  through  the  bodies  of  susceptible  animals.  The 
reverse  condition — that  is,  attenuation  of  the  virulence — 
may  be  effected  by  passing  the  microbes  through  relatively 
insusceptible  organisms.  Pasteur  in  this  way  attenuated 
the  bacillus  of  hog-erysipelas,  by  inoculating  rabbits  with 
successive  generations  of  the  organism.  In  the  same  way 
he  procured  a  mitigated  virus  of  hydrophobia  by  continued 
inoculation  of  monkeys. 

The  more  virulent  a  bacterium,  the  more  readily  does  it 
give  rise  to  infection,  and  the  more  severe  is  the  course  of  the 
latter.  With  a  small  amount  of  a  pneumococcus-culture, 
prepared  a  day  or  two  days  previously  from  an  infiltrated 
pneumonic  lung,  it  is  possible  to  destroy  rabbits  with  cer- 
tainty, in  from  twenty-four  to  forty-eight  hours  after  the 
development  of  symptoms  of  septicemia.  With  exactly  a 
like  amount  of  the  same  culture  it  is  possible,  two  or  three 
days  later,  to  induce  only  local  suppuration,  which,  after 
evacuation  of  the  abscess,  progresses  to  recovery ;  or  very 
slowly  and  insidiously,  though  without  septicemia,  to  death. 
After  a  further  interval  of  two  or  three  days,  inoculation 
practised  in  the  same  way  with  the  same  culture  is  unat- 
tended with  result — the  virulence  is  now  entirely  lost,  and 
infection  no  longer  takes  place.  If,  under  the  conditions 
named,  the  explanation  of  the  diminution  and  loss  of  viru- 
lence is  to  be  found  in  the  age  of  the  culture,  it  remains 
completely  concealed  under  other  circumstances.  Thus, 
there  may  be  present  in  the  same  membrane  in  a  given 
case  of  diphtheria,  intensely  virulent  diphtheria-bacilli,  and, 
besides,  others  free  from  all  virulence,  and  which,  therefore, 
are  designated  pseudodiphtheria-bacilli.  Inoculation  of  the 
former  is  capable  of  causing  spread  of  the  diphtheria,  while 
the  nonvirulent  bacilli  are  incapable  of  causing  the  spread 
of  the  disease. 

Finally,  a  further  illustration  from  human  pathology  may 
be  given  to  illustrate  the  relations  between  virulence  and 
infection.  For  the  acquisition  of  pneumonia  a  predispos- 
ing cause  frequently  is   necessary — a   lesion  of  the  lungs^ 


INFECTION.  39 

which  is  usually  induced  through  the  action  of  cold.  The 
disease  may  be  transmitted  also  from  one  pneumonic  pa- 
tient directly  through  the  sputum  to  other  individuals. 
Instances  of  house-epidemics  of  pneumonia  have  been  re- 
ported, and  which  are  capable  of  scarcely  any  other  expla- 
nation than  that  the  bacteria  were  present  normally  in  the 
upper  air-passages  of  the  individual  first  attacked,  and 
whence  they  have  gained  entrance  into  the  pulmonary  tis- 
sue, whose  resistance  has  been  diminished  in  consequence 
of  the  action  of  cold,  and  here  have  set  up  an  inflammatory 
process.  The  bacteria,  however,  that  are  obtained  from  a 
focus  of  disease  are  more  virulent  than  those  that  are  found 
upon  normal  skin  and  mucous  membrane.  The  virulence 
of  the  causative  bacteria,  which  is  responsible  for  the  in- 
tensity of  the  disease,  is  increased  in  turn  with  the  severity 
of  the  disease -process.  This  is  true  during  the  height  of 
the  infection  for  all  bacteria.  With  the  decline  of  the  dis- 
ease, naturally,  when  the  bacteria  have  remained  for  a  cer- 
tain time  in  the  organism  now  acquiring  immunity,  their 
virulence  lessens,  as  a  rule.  Returning  to  our  illustration 
of  pneumonia,  the  course  of  events  would  be  that  the 
pneumococci  derived  from  the  pneumonic  lung  of  the  indi- 
vidual first  affected  would  now  be  capable,  by  reason  of 
their  increased  virulence,  of  infecting  a  second  and  a  third 
individual  without  the  aid  of  predisposing  influences. 

A  striking  illustration  of  the  variation  in  virulence  of  mi- 
croorganisms within  the  living  body  is  furnished  also  by  the 
pyogenic  streptococci  and  staphylococci.  These  are  found 
in  association  with  all  possible  inflammatory  and  suppura- 
tive processes,  from  a  simple  panaris  to  the  most  intense 
septicemia  or  pyemia.  Their  morphologic  peculiarities  re- 
main the  same  throughout.  A  difference  is  apparent  only 
in  animal  experimentation.  The  cocci  obtained  from  the 
benign  affections  prove  slightly,  if  at  all,  virulent ;  while 
those  obtained  from  severe  infections  induce  the  most  dele- 
terious consequences  in  experimental  animals. 

(b)  The  Amount  and  the  Purity  of  the  Infectious 
Material. — To  induce  infection  experimentally  in  animals, 
a  definite  amount  of  the  culture  is  always  necessary,  and 
this  varies  for  different  bacteria  and  individual  species  of 
animals,  although  it  is  almost  constant  for  the  same  variety 
of  animals  and  of  bacteria.  If  a  smaller  amount  of  bacteria 
is    used,  infection  will    not   take    place.     This  relation    is 


40  CLINICAL  BACTERIOLOGY. 

most  evident  in  the  case  of  the  purely  toxic  bacteria. 
If  0.5  cu.  cm.  of  a  tetanus  bouillon-culture  of  deter- 
mined toxicity  are  necessary  to  cause  the  death  of  a  rab- 
bit, 0.3  cu.  cm.  may  perhaps  induce  passing  rigidity,  and 
the  introduction  of  o.  i  cu.  cm.  will  be  unattended  with  any 
result ;  and  if  o.ooooi  cu.  cm.  of  another  culture  will  cause 
the  death  of  a  white  mouse,  0.000005  ^^-  ^"^'  "^^7  possibly 
cause  transitory  mild  tetanus,  and  0.00000 1  cu.  cm.  no  dis- 
ease whatever.  The  greater  or  smaller  number  of  bacteria 
may  in  this  case  play  no  part  in  the  result,  but  the  larger 
amount  maybe  fatal  because  with  the  larger  number  of  bac- 
teria a  larger  amount  of  already  prepared  toxin  also  is  intro- 
duced; the  smaller  amount  of  toxin,  however,  which  the 
smaller  number  of  bacteria  carry  with  them,  is  readily  with- 
stood. With  the  infectious  bacteria,  also,  a  certain  amount 
of  the  infecting  material  is  necessary  to  induce  infection.  For 
dogs,  highly  virulent  pneumococci  injected  subcutaneously 
are  in  marked  degree  infectious  ;  they  multiply  rapidly,  giv- 
ing rise  to  extensive  inflammation  in  the  subcutaneous  tis- 
sues, and  they  cause  death — without  septicemia,  however. 
When  the  virulence  of  the  culture  is  sufficiently  intense, 
large  dogs  may  be  destroyed  by  as  little  as  0.5  cu.  cm.  of 
the  pneumococcus-culture.  There  thus  results  a  true  infec- 
tion, and  the  possibility  of  a  purely  toxic  action  may  safely  be 
excluded.  From  o.  I  to  0.3  cu.  cm.  of  the  same  culture, 
on  the  other  hand,  fail  to  induce  any  disease  whatever  in 
the  dog.  The  significance  of  the  amount  of  the  infecting 
material  is  more  questionable  in  the  case  of  those  bacteria 
that  give  rise  to  septicemia.  If  pneumococci,  which  readily 
cause  fatal  septicemia  in  rabbits,  are  introduced  into  the  cir- 
culation of  these  animals  in  great  dilution,  infection  does  not 
take  place.  On  the  other  hand,  according  to  some  observ- 
ers, in  the  case  of  the  septicemia  of  white  mice  and  of  an- 
thrax of  guinea-pigs,  a  single  bacillus,  presupposing  the 
virulence  of  the  culture  to  be  sufficiently  great,  may  be  ade- 
quate to  induce  infection.  This,  however,  appears  question- 
able, although  it  has  been  demonstrated  that  the  individual 
bacillus  also  in  these  two  instances  does  not  necessarily 
cause  infection,  and  that  even  one  or  two,  or  as  many  as  ten, 
of  the  most  virulent  anthrax -bacteria  of  the  guinea-pig  may 
be  borne  without  causing  appreciable  disease.  That  the 
amount  of  the  infecting  material  is  also  of  significance  in  the 
case  of  these  bacteria  endowed  with  especial  virulence  is 


INFECTION.  41 

shown  by  the  fact,  estabhshed  experimentally,  that  the 
larger  the  number  of  bacteria  introduced,  the  more  speedily 
does  the  death  of  the  animal  take  place. 

With  regard  to  the  purity  of  the  infecting  material,  the 
question  especially  of  mixed  infection — that  is,  infection  with 
a  mixture  of  bacteria — arises.  In  a  number  of  diseases  in 
man  several  varieties  of  bacteria  may  almost  always  be 
found  in  the  disease-focus ;  thus,  for  instance,  in  diphtheria 
streptococci  as  well  as  diphtheria-bacilli,  and  in  advanced 
tuberculosis  pyogenic  cocci  as  well  as  tubercle-bacilli.  The 
presence  of  one  variety  of  bacteria  may  facilitate  the  entrance 
and  the  activity  of  others,  whose  virulence  is  increased  by 
the  symbiosis  ;  in  other  words,  infection  with  the  one  variety 
of  bacteria  is  rendered  possible  through  the  agency  of  the 
other.  Attenuated  pyogenic  cocci  may  be  rendered  again 
virulent  by  the  simultaneous  introduction  of  bacterium  coli, 
of  proteus,  and  even  of  saprophytes,  such  as  prodigiosus,  or 
hay-bacillus.  Streptococci  are  said  to  restore  the  toxicity 
of  attenuated  diphtheria-bacilli  when  injected  simultaneously 
into  guinea-pigs.  The  causative  agents  of  typhoid  fever 
and  of  cholera  regain  their  infectiousness  when  they  are  in- 
troduced into  animals  in  association  with  streptococci,  coli 
commune,  or  with  metabolic  products  of  proteus.  Tetanus- 
bacilli  or  tetanus-spores  alone,  without  their  toxins,  do  not 
give  rise  to  disease,  as  has  been  demonstrated  experiment- 
ally by  Vaillard  ;  but  if  with  them  are  injected  other  bac- 
teria in  themselves  indifferent  (as  takes  place  in  natural  in- 
fection through  earth  and  splinters  of  wood),  germination 
takes  place,  with  toxin-production  and  the  development  of 
tetanus.  For  other  anaerobic  bacteria  similar  conditions 
appear  to  prevail ;  at  least,  it  is  possible  to  favor  materially 
infection  with  malignant  edema  and  symptomatic  anthrax 
by  simultaneous  inoculation  with  pyogenic  cocci,  proteus,  or 
prodigiosus,  or  their  metabolic  products.  With  such  mixed 
infection,  the  clinical  picture^ — the  infection — may  be  a 
mixed  one,  as  the  result  of  the  activity  of  the  various  bac- 
teria. Thus,  in  the  clinical  picture  of  septic  diphtheria  the 
distinctively  septic  symptoms  are  to  be  attributed  to  strepto- 
cocci ;  the  intermittent  fever  of  tuberculous  patients,  to  pyo- 
genic cocci.  In  other  cases,  however,  the  effect  of  the  activ- 
ity of  the  specific  bacterium  only  may  make  itself  manifest 
in  the  clinical  picture  (as  with  tetanus),  and  the  role  of  the 
second  is  exhausted  with  the  rendering  possible  of  infection. 


42  CLINICAL  BACTERIOLOGY. 

Finally,  it  is  also  possible,  as  Nencki  has  shown,  that  two 
microbes  may  produce  an  entirely  new  substance  through 
their  influence  upon  the  culture-medium,  and  which  neither 
of  the  two  bacteria  was  alone  capable  of  producing.  Also, 
the  observ'ation  of  Nencki's,  so  characteristic  for  the  signifi- 
cance of  mixed  infection,  is  to  be  borne  in  mind,  that  **  sterile 
solutions  of  grape-sugar,  simultaneously  injected  with  two 
given  bacteria,  are  much  more  rapidly  and  more  energetic- 
ally decomposed  than  by  either  of  the  two  germs  alone." 

(c)  The  Portals  of  Infection. — Natural  portals  of  in- 
fection are  constituted  by  all  those  parts  already  named 
that  communicate  with  the  external  world  (p.  36).  The 
most  important  infectious  agents  are  taken  up  with  the  in- 
spired air  and  with  the  nourishment  or  through  the  skin. 
The  uninjured  skin  forms  an  insuperable  barrier  that  can 
be  overcome  only  by  vigorous  inunction  of  bacteria  in  an 
ointment-basis.  It  is  possible,  in  this  way,  to  produce  fur- 
uncles by  the  rubbing  in  of  staphylococci,  and  general  infec- 
tion by  the  rubbing  in  of  anthrax -bacilli  or  of  glanders- 
bacilli.  If,  however,  a  breach  in  the  continuity  of  the  skin 
takes  place,  then  the  chances  for  the  invasion  of  bacteria  are 
rendered  much  more  favorable.  Superficial  cutaneous  fis- 
sures suffice  to  permit  the  bacteria  of  anthrax  and  of  septi- 
cemia to  gain  entrance  into  the  organism.  Deeper  subcuta- 
neous wounds  are  more  dangerous,  because  the  lax  tissues 
permit  absorption  in  much  greater .  degree.  Contused  and 
lacerated  wounds,  to  which  access  of  the  oxygen  of  the  air 
is  not  unobstructed,  favor  the  development  of  anaerobic 
bacteria,  especially  that  of  tetanus.  The  absence  of  oxygen 
appears,  further,  to  constitute  a  favoring  influence  for  the 
activity  of  the  ordinary  pyogenic  cocci.  Recent  wounds  take 
up  microorganisms  with  remarkable  rapidity  through  the 
opened  blood-vessels.  Within  as  short  a  time  as  thirty  or 
forty  minutes,  bacteria,  even  saprophytes,  placed  upon  a 
fresh  wound  may  be  found  within  the  internal  organs.  In 
the  case  of  old  suppurating  wounds,  on  the  other  hand, 
absorption  of  microorganisms  takes  place  only  in  quite 
limited  degree. 

The  mucous  membranes  also,  in  an  uninjured  state,  prove 
not  especially  susceptible  to  bacterial  invasion.  If,  how- 
ever, a  breach  in  the  continuity  of  the  epithelial  covering 
takes  place,  then  opportunity  is  afforded  for  the  entrance 
and  the  absorption  of  the  germs  present.      Exceptions  to 


INFECTION.  43 

the  rules  just  cited  are  furnished  by  a  number  of  mucous 
membranes  in  relation  to  certain  microorganisms.  The 
normal  conjunctiva  is,  for  instance,  susceptible  to  invasion  by 
the  gonococcus  and  also  by  the  bacillus  of  intestinal  diph- 
theria. Upon  the  mucous  membrane  of  the  urethra  like- 
wise, only  the  causative  agent  of  gonorrhea  thrives.  In  the 
mouth,  according  to  the  investigations  of  Sanarelli,  only 
two  microorganisms  develop  properly  :  the  diplococcus  of 
pneumonia  and  the  bacillus  of  diphtheria.  The  tonsils, 
however,  with  their  markedly  irregular  surface,  and  with 
their  richly  developed  lymphatic  structure,  do  not  share  in 
the  protection  of  the  remainder  of  the  mucous  membrane 
of  the  mouth,  but,  on  the  contrary,  constitute  a  frequent 
portal  of  entry  for  numerous  infections. 

The  gastric  juice,  by  reason  of  the  hydrochloric  acid  it 
contains,  is  disinfectant  and  bactericidal,  but  this  gastric- 
juice  hydrochloric-acid  barrier  has  for  a  long  time  been 
greatly  overestimated.  The  permanent  forms — the  spores 
— are  not  at  all  affected  by  the  gastric  juice,  and  the  resistant 
tubercle-bacilli  in  no  greater  degree  ;  and  even  less  resistant 
germs  pass  the  pylorus  so  rapidly,  especially  after  the 
ingestion  of  large  amounts  of  fluid,  that  the  gastric  juice 
is  not  afforded  sufficient  opportunity  to  cause  the  death 
of  the  microorganisms.  The  mucous  membrane  of  the 
intestine  is  far  more  markedly  predisposed  to  infection. 
The  cause  for  this  difference,  as  in  the  case  of  the  tonsils, 
must  be  looked  for  in  the  abundance  of  glands,  of  lymphatic 
elements,  and  of  the  absorptive  apparatus  generally. 

Bacteria  are,  under  certain  conditions,  absorbed  from  the 
mucous  membrane  of  the  air-passages,  and  are  then  in- 
tercepted by  the  bronchial  lymphatic  glands.  Only  in  this 
way  is  to  be  explained  the  not  uncommon  discovery  of  the 
presence  of  tuberculosis  of  these  glands,  with  complete 
immunity  of  the  lungs.  The  uterine  mucous  membrane  is, 
as  may  be  readily  understood,  a  most  suitable  surface  for 
the  absorption  of  infectious  agents  during  parturition  and 
also  during  menstruation. 

In  animal  experimentation  subcutaneous,  intravenous, 
and  intraperitoneal  injections  are  especially  employed. 
Other  methods  of  inoculation,  such  as  the  cutaneous,  the 
intraocular,  the  intracranial  (subdural),  etc.,  are  employed 
less  commonly,  and  only  for  special  purposes.  In  animal 
experimentation  the  same  amount  of  a  culture  exerts  differ- 


44  CLINICAL  BACTERIOLOGY. 

ent  effects  in  the  same  animal  in  accordance  with  the  site 
of  introduction.  An  amount  of  pneumococci  that,  injected 
subcutaneously,  would  cause  death  in  a  dog,  will,  when 
given  by  intraabdominal  injection,  cause  no  disturbance. 
Conversely,  cholera-bacilli  act  more  energetically  in  guinea- 
pigs  when  introduced  into  the  peritoneal  cavity  than  by 
subcutaneous  inoculation.  Cattle  tolerate  without  ill  result 
the  bacilli  of  symptomatic  anthrax  when  injected  into 
a  vein,  whereas  the  same  material  introduced  subcuta- 
neously invariably  gives  rise  to  disease.  In  the  same  way 
also  in  human  pathology  the  point  of  entrance  of  the  bac- 
teria into  the  body  is  of  importance  for  the  occurrence  of 
an  infection.  Thus,  cholera-infection  occurs,  as  a  rule,  only 
through  the  intestine,  pneumonic  infection  only  through  the 
upper  air-passages — at  least,  it  has  been  demonstrated  that 
the  subcutaneous  injection  of  not  too  large  amounts  of 
cholera-bacilli  or  of  pneumococci  is  without  injurious  effect 
upon  human  beings. 

(d)  The  Susceptibility  of  the  Infected  Organism  (Pre- 
disposition).— The  susceptibility  of  different  species  of 
animals  to  an  infectious  disease  varies  widely.  To  tetanus, 
for  instance,  the  guinea-pig  and  the  white  mouse  are  highly 
susceptible,  the  rabbit  far  less  so,  and  fowl  so  little  sus- 
ceptible that  it  is  only  with  difficulty  that  tetanus  can  be 
induced  in  these  animals.  To  no  variety  of  bacteria  are  all 
animals  equally  susceptible.  Thus,  while  cattle,  mice,  and 
guinea-pigs  are  highly  susceptible  to  anthrax,  rats,  dogs, 
and  birds  are  almost  entirely  insusceptible,  and  cold- 
blooded animals  tolerate  the  pathogenic  microorganisms 
almost  universally  without  injury. 

Also  in  the  same  animal  species  differences  in  suscepti- 
bility exist  toward  the  same  bacterium.  Thus,  field-mice 
suffer  from  glanders,  while  white  mice  do  not.  Older 
animals  are,  in  general,  less  readily  infected — that  is,  they 
are  less  susceptible  than  young  animals.  Congenital  sus- 
ceptibility is  designated  natural  predisposition.  This  pre- 
disposition is,  however,  not  constant  in  degree  even  in  the 
same  animal.  It  may  be  intensified  or  diminished.  Thus, 
insusceptible  animals  may  be  rendered  temporarily  sus- 
ceptible to  certain  diseases  by  protracted  hunger,  great 
muscular  exercise,  and  similar  influences.  Such  a  tempo- 
7'ary  predisposition  can,  for  instance,  be  induced  in  frogs  to 
anthrax  by  exposure  to  heat,  in  fowl  to  the  same  disease 


INFECTION.  45 

by  exposure  to  cold,  in  pigeons  by  hunger  or  long-con- 
tinued withholding  of  water,  and  in  white  mice  to  glanders 
by  the  production  of  phloridzin-diabetes.  In  the  same  way, 
intoxication  with  alcohol  or  with  various  substances,  espe- 
cially such  as  are  destructive  of  the  blood-corpuscles,  gives 
rise,  temporarily,  to  especial  susceptibility.  The  predispo- 
sition of  diabetics  to  certain  infections  (suppuration,  gan- 
grene, tuberculosis)  may  also  be  mentioned.  Likewise,  a 
temporary  predisposition  is  established,  according  to  the 
well-known  theory  of  Pettenkofer,  through  telluric  (ground- 
water elevation)  and  temporal  influences  (summer's  heat) 
when  an  epidemic  of  cholera  occurs. 

In  addition  to  the  general  predisposition  a  local  predis- 
position may  be  distinguished,  depending  upon  the  varying 
susceptibility  of  the  different  tissues  of  the  body.  Her- 
mann undertook  the  establishment  of  a  scale  of  susceptibility 
for  the  staphylococcus.  The  anterior  chamber  of  the'  eye 
proved  most  susceptible ;  then  followed  the  circulatory 
apparatus  of  the  rabbit ;  next  the  subcutaneous  connective 
tissue  of  the  dog ;  then  the  pleura,  the  cerebral  meninges, 
the  subcutaneous  tissue,  and  the  peritoneum  again  of 
the  rabbit.  Little  is  known  with  regard  to  the  actual  con- 
ditions upon  which  the  degree  of  predisposition  or  suscep- 
tibility of  a  body  for  a  given  species  of  bacteria  is  depend- 
ent. The  word  predisposition  is  only  an  expression  for 
the  sum  of  resistances  that  the  body  offers  to  infection. 
What  the  nature  of  these  resistances  is  will  be  fully  dis- 
cussed in  the  next  section  in  a  consideration  of  the  subject 
of  immunity. 

A  certain  measure  of  resistive  influences  against  infection 
must  be  present  in  every  animal  tissue  ;  at  least,  there  ap- 
pears to  be  no  absolute  susceptibility.  The  weapons  of 
the  bacteria  against  these  resistances  are  most  probably 
their  toxins;  in  this  way  the  significance  of  the  virulence 
and  of  the  amount  of  the  infectious  agents  introduced  is 
rendered  comprehensible.  On  the  other  hand,  it  must  be 
assumed  that  the  devices  mentioned  that  are  capable  of  in- 
creasing the  susceptibility  (inanition,  overexertion,  over- 
cooling  and  overheating,  anemia,  glycemia,  etc.)  diminish 
these  resistances  of  the  organism. 

With  regard  to  the  susceptibility  of  human  beings  to  bac- 
terial diseases,  this  is  comparatively  slight  for  most  infec- 
tious diseases — for  the  suppurations,  pneumonia,  cholera, 


46  CLINICAL   BACTERIOLOGY. 

typhoid  fever,  and  even  tuberculosis  ;  only  for  influenza,  for 
scarlet  fever,  and  especially  for  measles,  must  a  greater 
susceptibility  be  assumed  in  the  case  of  man.  From  the 
constant  contact  with  infective  bacteria  to  which  man  is 
continually  exposed,  infections  would  be  far  more  frequent 
than  they  really  are  if  the  predisposition  of  human  beings 
to  bacterial  diseases  were  not,  on  the  whole,  but  incon- 
siderable. 

In  general,  the  power  of  resistance  of  our  tissues  against 
bacteria  is  so  great  that,  for  infection  to  take  place,  an 
additional  special  contributing  cause  that  diminishes  this 
power  of  resistance — in  other  words,  a  predisposing  influ- 
ence— is  necessary.  Among  such  etiologic  factors  expo- 
sure to  cold,  traumatism,  emotional  disturbances,  and 
overexertion  (traumatic  pneumonia,  traumatic  tuberculosis, 
typhoid  fever  after  grief  and  worry,  etc.)  have  long  been 
known.  Further,  the  bacilli  may  gain  entrance  into  the 
body  in  especially  large  numbers,  or  they  may  possess  in- 
creased virulence,  as  is  the  case  in  the  event  of  direct  con- 
tagion, especially  in  times  of  epidemic,  when  the  bacteria 
possibly  have  several  times  completed  their  passage  through 
the  body. 

In  conclusion,  it  may  be  mentioned  that  the  infection  it- 
self may  act  as  a  predisposing  factor  in  the  development 
of  a  second  subsequent  infection.  When  one  variety  of 
bacteria  proliferates  in  a  body  in  which  another  variety  of 
bacteria  is  already  in  activity,  the  condition  is  designated  a 
secondary  infection.  Examples  are  afforded  by  some  varie- 
ties of  pneumonia  in  cases  of  typhoid  fever:  the  typhoid 
patient  is  attacked  by  the  pneumonic  infection  because  the 
resistance  of  his  tissues  is  impaired  as  a  result  of  the  influ- 
ence of  the  typhoid  virus.  Such  secondary  infections  play 
a  most  important  role  in  connection  with  the  infectious  dis- 
eases of  man.  The  oral  affections,  the  otitis,  the  broncho- 
pneumonia, the  cystitis,  the  suppurative  and  even  the  pyemic 
processes  that  so  frequently  complicate  the  primary  disease 
usually  are  merely  secondary  infections  ingrafted  upon  the 
original  disorder.  A  number  of  these  complications,  natu- 
rally, do  not  represent  true  secondary  infections,  but  only 
secondary  localizations  of  the  original  disease-causing  agent. 
Thus,  pneumonia  complicating  typhoid  fever  may  be  due 
to  typhoid-bacilli,  and  otitis  complicating  pneumonia  may 
be  due  to  pneumococci,  etc. 


INFECTION.  47 

Endemic  and  Epidemic  Occurrence  of  Infectious  Dis- 
eases.— Most  infectious  diseases  attack  human  beings  with 
varying  frequency.  We  speak  of  the  sporadic  occurrence 
of  a  disease  when  only  isolated  cases  appear ;  and  of 
endemic  distribution,  when  a  larger  number  of  cases  are 
constantly  present.  Measles,  scarlet  fever,  diphtheria,  and 
tuberculosis  are,  for  instance,  endemic  in  middle  European 
populations,  while  cerebrospinal  meningitis,  mumps,  etc., 
occur  only  sporadically.  Asiatic  cholera  prevails  endemic- 
ally  in  the  East  Indies. 

Under  special  conditions,  any  infectious  disease  may 
acquire  extraordinary  distribution,  attacking  a  much  larger 
proportion  of  the  population  than  before,  or  extending 
beyond  the  borders  of  its  previous  area  of  distribution,  and 
invading  neighboring  countries.  We  speak  of  epidemics 
of  typhoid  fever  and  of  diphtheria  when  the  usual  number 
of  cases  occurring  on  the  average  within  a  given  district  is 
considerably  exceeded.  At  certain  intervals  cholera  leaves 
its  Indian  home  to  invade  in  widespread  epidemics  {^pan- 
demics^ almost  the  entire  inhabited  world.  The  systematic 
causes  that  govern  the  occurrence  and  the  subsidence  of 
such  epidemics  have  been  made  the  subject  of  investigation, 
especially  by  Pettenkofer  and  by  Koch.  The  epidemic 
distribution  of  infectious  diseases  may  be,  in  large  part, 
explained  by  the  same  influences  that  are  responsible  for 
the  isolated  infection  ;  but  in  every  epidemic  the  special 
biologic  relations  of  the  causative  agents,  as  well  as  the 
varying  predispositions  of  human  beings,  demand  particular 
study.  Thus,  pandemics  of  influenza  are  readily  explicable 
from  the  fact  that,  on  the  one  hand,  the  bacilli  are  contained 
in  the  sputum  of  the  sick  and  with  this  are  carried  through 
the  air,  and,  on  the  other  hand,  the  susceptibility  of  human 
beings  to  this  infection  is  unusually  great.  For  the  com- 
prehension of  epidemics  of  cholera  it  is  important  to  know 
that  the  bacilli  are  contained  ifi  the  dejections,  and  that 
with  these  they  frequently  find  their  way  upon  linen,  cloth- 
ing, contaminated  articles  of  food,  etc.  ;  that  they  are 
conveyed  by  insects,  may  be  transported  with  soiled  mer- 
chandise, etc.  In  this  manner  extension  of  the  disease  may 
take  place  from  case  to  case  in  a  continuous  chain.  Explo- 
sive distribution  of  a  disease,  simultaneous  invasion  of  a 
large  part  of  a  community,  occurs  through  equable  distri- 
bution of  the  infective  material  over  an  extensive  area — as 


48  CLINICAL  BACTERIOLOGY. 

a  whole  city.  Such  dissemination  can  take  place  only 
through  the  air,  the  earth,  or  the  water,  which  are  com- 
mon to  all,  and  are  capable  of  acting  upon  the  entire  popu- 
lation of  a  city  at  the  same  time  and  in  the  same  way.  In 
this  connection  the  demonstration  made  by  Koch  in  recent 
epidemics  of  cholera  is  of  especial  significance — namely, 
that  the  cholera-dejections  gain  entrance  into  the  sewage 
through  the  first  unreported  cases  and  into  the  public 
waterways  through  those  living  along  the  streams  (sailors), 
and  from  both  of  these  sources  frequently  into  the  water- 
supply.  An  entire  community  may  thus  be  simultaneously 
infected  with  the  contaminated  drinking-water,  and  in  this 
way  a  cholera-explosion  takes  place. 

In  addition  to  these  more  special  causes,  social  conditions 
naturally  retain  their  significance  as  general  causes  of  epi- 
demics. An  impaired  state  of  nutrition  affecting  entire  classes 
in  a  community,  the  absence  of  air  and  of  light  in  dwell- 
ings, the  abuse  of  alcohol,  etc.,  must  naturally  increase  the 
predisposition  to  infection  in  the  same  way  as  lack  of  clean- 
liness and  density  of  population  multiply  the  possibility  of* 
contagion.  The  disease-germs  are,  further,  the  less  readily 
gotten  rid  of,  and  they  proliferate  the  more  freely  the  more 
filth  and  refuse  defile  the  surroundings  of  human  habita- 
tions. In  this  sense  contamination  of  the  soil  acts  as  an  im- 
portant cause,  and  purification  thereof  through  a  proper 
water-supply  and  drainage  as  a  fruitful  means  of  prophy- 
laxis, of  the  infectious  diseases.  The  fact,  also,  that  the 
warm  season  of  the  year  favors  the  growth  and  the  virulence 
of  the  causative  agents  of  disease,  and,  on  the  other  hand, 
through  the  numerous  digestive  derangements  resulting  in 
consequence  of  the  increased  heat  and  the  thirst,  increases 
the  predisposition  of  the  community,  may  be  mentioned  in 
further  explanation  of  the  occurrence  of  epidemics.  Thus, 
a  series  of  factors  may  be  traced  that  shed  light  upon  the 
previously  mysterious  origin  of  pestilences.  On  the  other 
hand,  it  must  be  emphasized  that  there  is  yet  much  that  is 
obscure  in  the  etiologic  relations  of  epidemics,  and  that 
further  investigation  is  necessary  to  clear  up  these  questions. 

The  Heredity  of  Infectious  Diseases. — The  transmis- 
sion to  the  offspring  of  chronic  disease  existing  in  either 
parent  at  the  time  of  conception  may  be  considered  as  direct 
infection  of  the  sperm-cell  or  the  ovum.  To  what  extent 
this  actually  takes  place  will  be  considered  in  discussing  the 


IMMUNITY,    IMMUNIZATION,   AND   CURE.  49 

heredity  of  tuberculosis  and  of  syphilis.  A  second  possi- 
bility is  that  the  predisposition  to  a  given  infection  is  in- 
herited by  the  child  from  its  parents.  This  aspect  of  the 
subject  also  will  be  more  fully  discussed  in  the  chapter  on 
Tuberculosis,  with  relation  to  which  alone  it  is  of  material 
importance. 

Intrauterine  infection  of  the  fetus  in  the  course  of  acute 
infectious  disease  in  the  mother  has  been  observed  re- 
peatedly. A  number  of  cases  are  on  record  in  which  chil- 
dren have  been  born  with  the  eruption  of  smallpox  or  with 
pneumonia.  As  the  result  of  numerous  experimental  in- 
quiries upon  this  subject — through  the  infection  of  pregnant 
animals — it  may  be  accepted  that  the  healthy  placenta  con- 
stitutes a  dense  filter,  which  permits  the  passage  only  of  the 
toxins,  but  never  of  the  bacteria,  circulating  in  the  blood  of 
the  mother.  Living  disease-germs  can  pass  over  to  the 
fetus  only  when  a  lesion  of  the  placenta  is  induced  through 
slight  hemorrhages  or  in  some  other  way.  More  common 
than  intrauterine  infection  is  infection  during  birth,  of  which 
the  blennorrhea  of  the  new-born  is  a  conspicuous  example. 


EL  IMMUNITY,  IMMUNIZATION,  AND  CURE. 

Immunity  is  the  insusceptibility  to  an  infectious  disease, 
the  slighter  tendency  of  an  organism,  or  the  complete  im- 
possibility, to  be  attacked  by  this  disease.  This  property 
may  be  congenital  in  animals  and  man  as  natural  immunity. 
Our  domestic  animals  are  never  attacked  by  the  acute  exan- 
themata so* widely  disseminated  among  human  beings  ;  and 
birds  under  natural  conditions  never  suffer  from  anthrax,, 
which  is  not  at  all  uncommon  among  cattle.  In  the 
severest  epidemics  of  cholera  a  large  number  of  persons  es- 
cape the  disease,  including  even  some  living  under  the  most 
unhygienic  conditions  and  without  any  special  precaution- 
ary measures,  while  those  by  whom  they  are  surrounded 
are  attacked  without  exception.  Under  these  and  all  like 
conditions  a  natural  protection  against  the  given  disease 
exists  :  the  individuals  possess  a  congenital,  natural  immu- 
nity— that  is,  the  infectious  agents  that  have  gained  entrance 
into  the  organism  are  incapable  of  inducing  the  specific  dis- 
ease-manifestations to  which  they  give  rise  in  other  non- 
immune organisms. 
4 


60  CLINICAL  BACTERIOLOGY. 

In  the  majority  of  cases  the  protection  that  exists  is 
against  the  Hving  causes  of  disease  themselves,  and  which 
are  incapable  of  development  within  the  body  of  the  particu- 
lar animal.  Much  more  rarely  the  basis  of  natural  immu- 
nity consists  in  an  insusceptibility  of  the  organism  to  bacterial 
(p.  29)  or  similar  poisons  (snake-venom,  ricin,  abrin).  As 
examples  of  this  so-called  natural  immunity  to  poisons  may 
be  mentioned  the  rather  marked  immunity  of  fowl  to  tetanus- 
toxin,  of  rats  to  diphtheria-toxin,  and  of  swine  to  snake- 
venom.  Also  in  the  case  of  the  most  marked  immunity  to 
poisons  the  importance  of  destruction  of  the  microorganisms 
responsible  for  the  poisons  is  not  to  be  underestimated. 

In  contradistinction  from  natural  immunity  is  acquired 
immmiity.  Human  beings  are  attacked  but  once  by  a 
number  of  infectious  diseases — a  manifestation  that  is  most 
marked  in  the  case  of  scarlet  fever,  of  measles,  and  of 
smallpox,  but  which  is  distinctly  observable  also  in  the  case 
of  cholera,  typhoid  fever,  etc.  Recovery  from  the  disease 
has,  under  these  conditions,  conferred  immunity — a  state  of 
protection  against  the  same  disease.  All  efforts  to  estab- 
lish an  artificial  immunity  through  active  intervention  are 
based  upon  this  natural  process.  The  act  through  which 
this  end  is  attempted  is  designated  immunization  or  vacci- 
nation. The  immunity  effected — the  disease-protection — is 
also  designated  protective  inoculation. 

The  oldest  method  of  immunization  is  represented  by 
vaccination  for  smallpox.  Recovery  from  mild  vaccine- 
disease  protects  against  severe  variolous  infection.  The 
causative  agent  of  smallpox  is  as  yet  not  known,  but,  from 
all  else  that  is  known  concerning  immunity  and  immuniza- 
tion, it  may  be  inferred  that  the  causative  agent  of  cow- 
pox  is  identical  with  that  of  variola,  and  represents  an 
attenuated  form  of  the  latter — a  view  that  has  an  experi- 
mental basis,  especially  from  the  investigations  of  Fischer 
(Karlsruhe). 

In  experimental  pathology  it  is  now  possible  to  immu- 
nize animals  against  many,  if  not  most,  infectious  diseases 
whose  causative  agents  are  known.  Progress  in  this  direc- 
tion dates  from  the  inoculation  against  anthrax  undertaken 
by  Pasteur,  who  attenuated  anthrax-bacilli  by  exposure  to 
high  temperatures,  and  in  this  way  obtained  two  vaccines. 
Vaccine  I  (exposure  for  from  fifteen  to  twenty  days  at  a 
temperature  of  42°  or  43°  C. — 107.6°  or  109.4°  F.)  pro- 


IMMUNITY,    IMMUNIZATION,  AND   CURE.  51 

tected  against  vaccine  II  (exposure  for  from  ten  to  twelve 
days  at  42°  or  43°  C. — 107.6°  or  109.4°  F.),  and  prelim- 
inary treatment  with  the  latter  afforded  protection  against 
virulent  anthrax.  The  process  here  is  quite  analogous 
to  that  of  vaccination  for  smallpox.  Vaccine  I  induces  the 
mildest  degree  of  anthrax,  recovery  from  which  confers 
upon  the  organism  the  ability  to  withstand  the  moderate 
infection  with  vaccine  II,  and  this  in  turn  confers  protec- 
tion against  the  severest  form — against  true  anthrax.  The 
important  and  novel  feature  in  this  method  of  immunization 
consists  in  the  application  of  heat  to  attenuate  the  causative 
agent  of  the  disease.  That  which  is  effected  by  passage 
through  the  body  of  the  cow  in  the  case  of  cowpox-inocu- 
lation,  the  reduction  of  the  virulence  of  the  disease-virus,  is 
effected  similarly  by  heat. 

As  the  possibility  of  immunization  through  heated  bac- 
terial cultures  has  been  demonstrated  by  numerous  later 
experiments  for  various  other  infections,  there  can  no  longer 
be  any  doubt  as  to  the  general  applicability  and  the  legiti- 
macy of  this  procedure.  The  rule  in  question  may  be  thus 
expressed  :  Above  the  temperature-optimum — that  is,  that 
temperature  at  which  a  bacterium  thrives  best  and  is  most 
virulent — ^between  this  optimum  and  that  temperature  that 
causes  death  of  the  bacterium,  there  is  for  every  variety  of 
bacteria  a  temperature  at  which,  though  it  still  lives,  it 
loses  in  virulence.  This  attenuation-temperature  is  for 
most  bacteria  between  40°  C.  (104°  F.)  and  70°  C.  (158° 
F.).  The  higher  the  temperature  selected,  the  closer  to  the 
fatal  temperature,  the  more  rapidly  does  the  attenuation 
take  place.  The  property  thus  newly  acquired  is,  however, 
generally  not  constant,  the  bacteria  soon  returning  in  suc- 
ceeding generations  to  their  original  virulence.  True  vac- 
cines— that  is,  varieties  of  bacteria  that  remain  attenuated 
constantly  in  all  successive  daughter-cultures — are  obtained 
only  by  means  of  the  lowest  possible  temperature.  Thus, 
anthrax-bacilli  are  materially  attenuated  on  exposure  to  a 
temperature  of  55°  C.  (131°  F.)  in  ten  minutes,  and  at  a 
temperature  of  42.6°  C.  (108.7°  F.)  in  not  less  than  from 
fourteen  to  twenty  days  ;  but  the  latter  only  are  available 
as  vaccines.  When,  however,  all  that  is  desired  is  the  pro- 
duction from  a  bouillon-culture  of  an  immunizing  fluid  for 
temporary  use,  the  attenuation-temperature  is  made  as  high 
as  possible ;  inasmuch  as  the  cultivation  of  true  vaccines  in 


52  CLINICAL  BACTERIOLOGY. 

the  case  of  the  more  parasitic  varieties,  which  spontaneously 
lose  their  virulence  gradually  in  culture,  has  not  hitherto 
proved  successful.  The  introduction  of  a  living  bacterial 
culture  attenuated  by  means  of  such  a  degree  of  tempera- 
ture confers  immunity  to  the  corresponding  bacterial  disease. 
The  act  of  immunization  induces  a  mild  disease,  recovery 
from  which  gives  rise  to  the  immunity.  For  this  reason  the 
advent  of  immunity  is,  under  these  circumstances,  never  im- 
mediate, but  it  takes  place  only  after  the  termination  of  the 
mild  disease  induced.  According  to  the  degree  of  atten- 
uation, and  the  amount  of  bacteria  introduced,  as  well  as 
the  mode  of  introduction,  the  time  that  elapses  before  im- 
munization is  complete  varies  from  three  to  fourteen  days. 

The  role  of  heat  in  the  attenuation  of  cultures  employed 
for  immunization  may  be  assumed  by  a  number  of  other 
factors.  Thus,  immunization  has  been  practised  with  cul- 
tures through  which  electric  currents  have  been  passed 
for  some  time ;  further,  with  cultures  that  have  been  ex- 
posed to  sunlight ;  but  most  commonly  with  cultures  that 
have  been  exposed  to  the  action  of  chemic  substances  (anti- 
septics). Of  the  large  number  of  these  substances  and 
methods  of  immunization,  we  shall  mention  only  the 
attenuation  of  anthrax-bacilli  by  the  addition  of  potassium 
bichromate  or  of  carbolic  acid ;  further,  immunization 
to  diphtheria  and  to  tetanus  by  means  of  cultures  to 
which  iodin  trichlorid  has  been  added ;  and  immunization 
by  means  of  thymus-bouillon-cultures,  in  which  the  cellular 
substances  of  the  thymus  gland  are  supposed  to  exercise 
the  attenuating  influence. 

Finally,  the  living  organism  also  possesses  under  certain 
circumstances  the  capability  of  diminishing  the  virulence 
of  microorganisms.  Pasteur  demonstrated  this  first  for  the 
bacillus  of  swine-erysipelas,  by  passing  this  organism 
repeatedly  through  the  bodies  of  rabbits,  which  are  but 
slightly  susceptible.  According  to  this  method,  two  vac- 
cines against  swine-erysipelas  of  different  strength  are 
obtained,  which  are  employed  largely  and  successfully  in 
France.  Pasteur  also  obtained  a  marked  reduction  in  the 
virulence  of  the  virus  of  hydrophobia  through  continued 
inoculation  of  monkeys.  Cowpox,  as  has  already  been 
stated,  represents  likewise  only  an  attenuated  modification 
of  smallpox,  the  attenuation  being  effected  by  the  passage 
of  the  disease  through  cattle. 


IMMUNITY,   IMMUNIZATION,   AND   CURE.  53 

In  place  of  attenuated  causative  agents  of  disease,  quan- 
tities insufficient  to  cause  infection  may  be  introduced. 
Immunity  has  been  repeatedly  induced  by  marked  dilution 
of  unaltered  living  cultures.  The  small  number  of  micro- 
organisms introduced  causes  only  slight  local  disease,  in 
the  sequence  of  which  a  certain  degree  of  immunity  be- 
comes manifest. 

An  essential  difference  exists  between  the  methods  of 
immunity  hitherto  discussed  and  those  in  which  the  im- 
munizing material  no  longer  contains  living  bacteria.  With 
the  germ-free  filtrate  of  a  bacterial  culture  it  is  possi- 
ble, in  many  cases,  in  an  analogous  manner  to  that  with 
the  attenuated  living  bacteria,  to  confer  immunity,  and  the 
attenuated  virus  (through  heat,  chemic  substances,  etc.),  as 
well  as  the  unchanged  fully  virulent  filtrate,  may  be  em- 
ployed ;  the  latter,  naturally,  in  a  dilution  and  in  amount 
below  the  lethal.  The  amount  of  toxin  is  gradually  in- 
creased, a  sufficiently  long  interval  being  permitted  to 
elapse  after  the  introduction  of  the  next  higher  dose  for 
the  animal  to  recover  completely  from  the  action  of  the 
previous  inoculation,  and  to  regain  its  original  weight.  It 
may  be  assumed  that  here  also  recovery  from  the  disease 
coincides  with  the  establishment  of  immunity,  and  this 
seems  to  point  to  the  fact  that  not  the  bacteria  themselves, 
but  rather  the  materials  produced  by  them,  contribute  to 
the  immunizing  effect  of  the  disease. 

Such  methods  of  immunization  as  seek  to  confer  immu- 
nity by  means  of  the  metabolic  products  of  bacteria,  and 
which  succeed  in  most  cases,  have  been  proposed  in  large 
number.  Besides  the  germ -free  filtrate  of  bacterial  cultures, 
bacterial  poisons  of  a  most  varied  source  have  been  em- 
ployed. Immunization  to  cholera-bacilli  and  to  typhoid- 
bacilli,  for  instance,  has  been  attempted  by  R.  Pfeiffer  with 
toxic  substances  that  are  contained  within  the  bodies  of  the 
bacteria  themselves.  Similarly,  Koch,  by  mechanical  tri- 
turation and  repeated  centrifugation,  obtained  from  the 
bacilli  themselves  tuberculin  R,  with  which,  according  to 
his  own  statement,  he  was  able  to  protect  guinea-pigs 
against  highly  virulent  tubercle-bacilli.  Immunization  with 
bacterial  metabolic  products  has  acquired  the  greatest  im- 
portance, however,  in  the  cases  of  diphtheria  and  tetanus. 
This  fact  is  not  surprising,  as  both  of  these  diseases 
belong  to  the  toxic  infections  in  which  general  symptoms 


54     •  CLINICAL  BACTERIOLOGY. 

of  intoxication  especially  predominate  over  those  due  to  the 
local  effects  of  the  bacterial  irritants. 

We  have  thus  far  considered  two  modes  of  immunization 
— (i)  the  attenuation-method  (Pasteur),  in  which  attenuated 
cultures  of  living  bacteria  or  attenuated  toxins  are  employed  ; 
and  (2)  the  dilution-method,  in  which  minimal  amounts  of 
virulent  cultures  or  toxic  metabolic  products  are  employed. 

Immunity  to  certain  microorganisms  may  further  be  con- 
ferred by  introducing  them  into  the  organism  to  be  vacci- 
nated through  a  portal  of  entry  that  is  different  from  that 
through  which  the  same  bacteria  gain  entrance  under 
natural  conditions.  On  introducing  the  virus  of  pleuro- 
pneumonia of  cattle  into  the  extremity  of  the  tail,  there 
results,  for  instance,  insignificant  local  disease,  which  is 
followed  by  immunity.  The  subcutaneous  injection  of 
cholera-cultures  for  the  purpose  of  conferring  immunity  to 
Asiatic  cholera  may  also  be  mentioned. 

More  difficult  of  comprehension  is  a  smaller  number  of 
methods  of  immunization,  in  which,  apparently,  the  bacteria 
themselves  take  no  part.  Thus,  for  instance,  injections  of 
hydrogen  dioxid  are  said  to  afford  protection  against  sub- 
sequent infection  with  diphtheria-bacilli.  -Under  these  cir- 
cumstances it  is  to  be  assumed  that  either  the  previously 
injected  material  remains  at  the  seat  of  injection,  where  it 
encounters  the  bacilli  subsequently  introduced,  or  their 
poisons,  and  these  are,  to  a  certain  degree,  thereby  at- 
tenuated within  the  body ;  or  there  may  result  an  intensifi- 
cation of  those  forces  of  the  organism  that  oppose  barriers 
to  the  infection — forces  that,  as  has  already  been  said,  are 
present  also  in  the  most  susceptible  organism,  at  least  in  a 
rudimentary  degree.  But  few  immunizing  methods  of  this 
kind  are  known,  and  they  are  of  little  significance  as  com- 
pared with  the  large  number  of  methods  already  mentioned, 
in  which  the  bacteria  themselves,  or  their  metabolic  pro- 
ducts, participate  in  the  establishment  of  immunity. 

The  last  method  of  immunization  to  be  considered  is 
that  suggested  by  Richet  and  Hericourt,  and  devised  by 
Behring  and  Kitasato — namely,  i7mnunization  by  means  of  the 
blood-serum  of  immunized  animals.  Though  demonstrated 
by  the  investigators  named  as  useful  only  for  staphylo- 
coccous  septicemia,  as  well  as  for  diphtheria  and  for  tetanus, 
this  method  was  subsequently  employed  against  the  infec- 
tions due  to  other  bacteria,  and  found  to  be  practicable. 


IMMUNITY,   IMMUNIZATION,  AND   CURE.  55 

The  blood-serum  of  an  animal  that  has  been  immunized 
according  to  one  or  the  other  of  the  methods  already  men- 
tioned is  capable  of  conferring  immunity  upon  a  susceptible 
animal  not  previously  treated.  The  same  property  that 
belongs  to  the  blood-serum  is  possessed  by  all  of  the  tissue- 
fluids,  as  well  as  the  milk  and  the  egg-yolk,  of  highly  im- 
munized animals.  The  most  significant  feature  of  this 
method  of  immunization  is  the  simplicity  and  the  rapidity 
with  which  it  confers  immunity.  The  immunity  is,  appar- 
ently, induced  immediately  as  a  result  of  the  serum-injection, 
and  no  such  phenomenon  is  observed  as  recovery  from  a 
disease  marking  the  development  of  immunity.  On  the 
other  hand,  the  immunity  conferred  by  the  injection  of 
serum  lasts  only  a  few  weeks,  and  is  thus  of  much  shorter 
duration  than  that  induced  more  slowly  through  bacterial 
activity.  Ehrlich  has  designated  as  passive  the  immuniza- 
tion that  is  effected  by  means  of  serum  and  in  which  the 
affected  organism  passes  through  no  disease,  in  contradis- 
tinction from  all  other  active  methods  of  immunization  in 
which  a  more  or  less  severe  disease  must  be  passed  through. 
Immunization  by  means  of  serum  thus  is  mediate  {indirect), 
in  contrast  with  immediate  {direct)  immunization  by  means 
of  bacteria  and  their  products.  To  what  extent  we  have 
gained  a  comprehension  of  the  processes  that  take  place 
in  the  act  of  immunization  with  serum  will  be  discussed 
later.  Here  may  be  mentioned  only  the  one  direction  in 
which  this  method  of  immunization  has  attained  some 
degree  of  importance  :  it  permits,  in  certain  cases,  the  appli- 
cation of  a  harmless  test  as  to  the  existence  of  immunity. 
Whereas  formerly  this  could  only  be  determined  by  induc- 
ing infection,  at  present  a  small  amount  of  blood  is  obtained 
from  the  individual  under  examination,  and  observation  is 
made  to  determine  if  the  serum  is  capable  of  conferring 
immunity  upon  a  susceptible  animal.  In  this  way  it  has 
become  possible  to  decide  with  experimental  certainty  as 
to  the  existence  or  not  of  immunity  also  in  human  beings. 
It  must  not,  however,  be  forgotten  that  the  blood-serum  is 
by  no  means  always  an  index  of  the  presence  or  absence 
of  immunity.  Behring  refers  to  horses  highly  protected 
against  tetanus,  whose  blood-serum  exhibited  no  protective 
activity.  Upon  the  other  hand,  there  are  animals  whose 
serum  possesses  marked  immunizing  properties,  and  which 
themselves  succumb  to  the  mildest  infection.     Metschnikoff 


66  CLINICAL  BACTERIOLOGY. 

found  in  blood-serum  from  cholera-cadavers,  in  a  number 
of  cases,  a  protective  influence  that  at  times  was  wanting 
in  the  blood  of  convalescents.  We  shall  return  to  this 
variation  in  the  relations  between  existing  immunity  and 
the  immunizing  power  of  the  blood-serum. 

Quantitative  Limitations  of  Immunity. — Immunity  is 
always  relative ;  absolute  immunity  is  theoretically  incon- 
ceivable. The  susceptibility  or  the  insusceptibility  to  dis- 
ease, on  the  one  hand,  and  the  intensity  of  the  disease,  upon 
the  other  hand,  are  more  or  less  marked.  All  processes  in 
this  connection  stand  in  regular  quantitative  dependence 
upon  one  another.  A  mathematically  accurate  measure  of 
these  variations  in  degree  is  at  present  impossible,  as  the 
bacterial  poisons  have  thus  far  eluded  chemic  analysis,  and 
a  unit  of  measure  from  which  to  start  out,  therefore,  does 
not  yet  exist.  Approximately  correct  investigations  have, 
however,  been  made  more  particularly  in  the  case  of 
tetanus  and  of  diphtheria,  whose  bacterial  cultures  contain 
powerful  poisons  that  pass  over  into  the  germ-free  filtrate 
of  bouillon-cultures.  It  has  been  shown  in  this  connection 
that  a  definite  amount  of  the  immunizing  culture  establishes 
a  definite  degree  of  immunity,  that  with  further  introduction 
of  the  immunizing  substances  the  degree  of  immunity  also 
increases,  and  finally  that  the  immunizing  power  of  the 
blood-serum  increases  to  a  certain  degree  with  the  degree 
of  the  existing  immunity.  It  is  obvious  that  the  pro- 
tection conferred  by  inoculation  is  never  absolute.  A  de- 
gree of  immunity,  however  high,  can  protect  only  against 
a  definite  degree  of  disease  ;  if  the  highly  immune  animal  is 
infected  with  an  amount  of  bacteria  or  of  toxin  representing 
a  still  higher  degree  of  disease,  it  will  finally  succumb. 
The  highest  degree  of  immunity  that  is  known  is  probably 
that  possessed  by  fowl  to  tetanus,  which  sufficiently  pro- 
tects against  all  danger  of  infection  that  exists  in  nature ; 
there  is  no  natural  tetanus  among  fowl.  Even  this  high 
degree  of  immunity  may,  however,  be  unable  to  withstand 
an  excessive  degree  of  intoxication  in  laboratory -experimen- 
tation, and,  in  fact,  tetanus  has  thus  been  developed  in  a 
typical  manner  in  fowl  and  pigeons. 

Specificity  of  the  Immunity. — Immunity  is,  in  general, 
specifically  limited.  The  immunity  conferred  by  vaccina- 
tion protects  only  against  smallpox ;  that  which  follows 
recovery  from  an  attack  of  scarlet  fever  does  not  protect 


IMMUNITY,   I  AjO^NlZATE»^'  AND*  qUREy^       1   57 

against  measles.  In  the  same  way  itTias  'been  demonstrated, 
also  experimentally,  that  the  immunity  established  by  the 
various  methods  of  immunization  is  specifically  limited. 
Preliminary  treatment  with  pneumococcus-cultures  protects 
only  against  pneumococcus-infection ;  inoculation  with 
typhoid-bacilli  protects  only  against  infection  with  these. 
Also,  in  the  transmission  of  immunity  through  blood-serum 
a  specific  limitation  of  the  activity  of  the  serum  has  been 
demonstrable  in  all  of  the  cases  examined.  It  is  noteworthy 
that  when  an  animal  is  protected  against  several  infections 
through  a  combination  of  methods  of  immunization,  its 
blood-serum  likewise  is  capable  of  conferring  protection 
against  all  of  these  infections.  Specificity  of  immunity  and 
of  immunization  must  be  considered  the  rule,  to  which, 
however,  there  appear  to  be  exceptions.  Thus,  Roux  re- 
ported at  the  International  Hygienic  Congress  at  Budapest 
that  tetanus-serum  is  antitoxic  not  only  to  tetanus-toxin, 
but  also  to  snake-venom,  while  snake-venom-serum  in  turn 
proves  active  against  scorpion-poison. 

Heredity  of  Immunity. — Immunity  is  hereditary :  it 
passes  from  mother  to  child — that  is,  it  is  conveyed  with 
the  blood  from  the  mother  to  the  child.  In  addition,  as 
Ehrlich's  experiments  have  shown,  immunity  is  transmitted 
through  the  milk  of  immune  mothers,  so  that  the  immunity 
established  by  nursing  fortifies  that  which  is  inherited.  The 
immunizing  property  of  milk  corresponds  with  the  immu- 
nizing power  of  the  egg -yolk  of  immunized  fowl. 

Inherited  immunity  is  naturally  only  transitory,  lasting 
but  a  few  weeks,  and  ceasing  with  the  elimination  from  the 
organism  of  the  infant  of  the  antitoxins  conveyed  through 
the  blood  during  fetal  life,  and  through  the  milk  during  the 
period  of  lactation  (p.  70).  The  conveyance  of  antitoxins 
through  the  milk  during  the  period  of  lactation  has  been 
demonstrated  by  Ehrlich  for  ricin,  abrin,  and  tetanus  in  the 
case  of  mice.  Vaillard  has  been  able  to  confirm  the  accuracy 
of  these  observations.  He  has,  however,  shown  further 
that  they  are  by  no  means  universally  applicable,  and  that 
they  are  not  to  be  applied  without  qualification  to  all 
species  of  animals. 

Investigations  into  the  Causes  of  Immunity. — Of  the 
older  theories  as  to  the  causes  of  immunity  only  the  reten- 
tion-theory and  the  exhaustion- theory  need  be  mentioned. 
According  to  the  first,  there  exists  a  substance  that  the 


68  CLINICAL  BACTERIOLOGY. 

disease  leaves  behind  in  the  body,  and  that  prevents  re- 
peated development  of  the  same  disease-causing  agents  in 
the  organism.  According  to  the  second,  recovery  from  a 
disease  is  attended  with  the  consumption  of  some  substance, 
without  the  presence  of  which  the  bacteria  are  incapable  of 
existence  within  the  body.  Probably  neither  of  these  hy- 
potheses in  its  original  form  has  advocates  at  present.  For 
a  considerable  time  the  scientific  world  was  dominated  almost 
exclusively  by  the  Phagocytic  Theory  of  Metschnikoff. 
According  to  this,  the  wandering  cells,  especially  the  white 
blood-corpuscles,  of  the  immune  and  the  immunized  organ- 
ism, as  phagocytes  take  up  the  invading  bacteria,  prevent 
their  germination,  and  the  production  of  toxins,  and,  finally, 
cause  their  destruction.  In  susceptible  organisms,  on  the 
other  hand,  the  bacteria  are  not  attacked,  multiply,  and  gen- 
erate their  poisons ;  and  when  they  gain  entrance  into 
white  blood-corpuscles,  they  prove  victorious  in  the  con- 
flict that  takes  place  in  every  instance  between  the  phago- 
cytes and  the  bacteria,  and  they  destroy  the  leukocytes. 
There  are  thus  not  only  mobile  phagocytes — as  such  are 
known  the  wandering  cells,  the  mononuclear  and  multi- 
nuclear  leukocytes,  with  the  exception  of  the  small  lymph- 
ocytes and  the  mast-cells — but  ^}iSO  fixed  phagocytes,  which 
are  represented  by  endothelial  cells,  Kupffer's  stellate  cells, 
the  pulp-cells  of  the  spleen  and  of  the  bone-marrow,  and 
connective-tissue  cells.  Metschnikoff  designates  the  poly- 
nuclear  leukocytes  and  wandering  cells  microphages ;  the 
large  mononuclear  leukocytes  and  the  fixed  phagocytes, 
macrophages.  The  principal  role,  however,  is  played  by 
the  mobile  phagocytes,  which,  in  the  sequence  of  infection, 
appear  in  large  numbers  upon  the  scene  of  conflict.  The 
hastening  of  the  leukocytes  to  the  threatened  area  depends 
upon  the  fact  that  in  the  immune  body  the  bacteria  generate 
certain  substances  that  attract  the  white  blood-corpuscles, 
and  that,  since  Pfeiffer's  investigations,  are  said  to  exert 
positive  chemotaxis.  If,  however,  the  leukocytes  are  re- 
pelled, the  condition  is  designated  negative  chemotaxis. 

The  phagocytic  doctrine  is  based  upon  an  exceedingly 
large  number  of  most  careful  observations.  It  is  undeni- 
ably correct  that  the  phagocytic  process  occurs  most  con- 
spicuously in  those  cases  that  terminate  favorably — that  is, 
in  the  conquest  of  the  microorganisms  by  the  individual 
organism — and  that,  on  the  other  hand,  it  is  less  marked 


IMMUNITY,   IMMUNIZATION,  AND   CURE.  59 

when  the  bacteria  gain  dominance.  That  the  leukocytes 
not  only  incorporate  the  bodies  of  dead  bacteria,  but  also 
take  up  living  microbes,  to  destroy  them  later,  can,  like- 
wise, no  longer  be  doubted,  since  Metschnikoff  observed  in 
the  hanging  drop  how  anthrax-bacilli,  already  inclosed 
within  phagocytes,  still  developed,  multiplied,  and  produced 
virulent  cultures.  It  is  possible,  also,  that  a  number  of 
methods  of  immunization,  in  which  relatively  simple  pro- 
tective fluids  are  employed,  induce  their  effects  especially 
through  phagocytosis.  Thus,  for  instance,  intraperitoneal 
injection  of  normal  blood-serum,  of  physiologic  salt-solu- 
tion, of  bouillon  and  other  substances,  by  means  of  which 
it  is  possible  to  protect  guinea-pigs  against  an  otherwise 
lethal  injection  of  cholera-vibrios,  are  powerful  stimulants 
of  phagocytosis. 

In  the  interpretation  of  his  observations  Metschnikoff 
has,  however,  gone  altogether  too  far.  The  inflammatory 
local  reaction  is,  in  the  case  of  infections  that  pursue  a  favor- 
able course,  present,  it  is  true,  and  prognostic  conclusions 
may  even  be  formulated  from  its  occurrence  ;  but  besides 
phagocytosis — and  in  this  the  opponents  of  Metschnikoff 
must  be  conceded  to  be  correct — other  factors  enter  into 
play.  Among  these  are  the  bactericidal  or  antidotal  prop- 
erties that  can  be  demonstrated  to  be  present  in  the  tissue- 
fluids,  especially  in  the  blood-serum  of  immune,  and,  in 
greater  degree  still,  of  immunized  animals.  These  are  to 
be  attributed  to  a  number  of  substances  that,  although  thus 
far  not  chemically  defined,  are  distinctly  differentiable  ac- 
cording to  their  physiologic  activities.  These  are  appro- 
priately grouped  together  as  Anti-bodies.  They  may  be 
divided  into  (i)  bactericidal,  (2)  lysogenic,  (3)  agglutinating, 
and  (4)  antitoxic  substances. 

I.  Bactericidal  Substances. — The  property  of  the 
bodily  fluids  to  destroy  bacteria  and  inhibit  their  activity 
was  first  observed  when  microorganisms — as  a  rule  anthrax- 
bacilli — were  introduced  into  immune  animals,  and  the  bac- 
teria were,  after  a  time,  seen  to  undergo  degeneration  at 
the  site  of  inoculation.  To  establish  the  certainty  that  the 
living  cells  did  not  participate  in  this  process,  the  anthrax- 
bacilli  were  introduced  inclosed  in  paper  bags  and  the  like. 
In  the  experiments  thus  conducted  various  investigators 
observed  degeneration  of  the  inoculated  anthrax-bacilli, 
and  in  some  instances  even   destruction  of  their  spores. 


60  CLINICAL   BACTERIOLOGY. 

Metschnikoff  and  his  pupils,  however,  obtained  opposite 
results,  finding  that  under  the  conditions  named  anthrax- 
spores  thrive  and  produce  virulent  cultures. 

Subsequently,  the  antibacterial  activity  of  the  bodily 
fluids,  especially  the  blood-serum,  was  studied  in  the  test- 
tube.  The  investigations  of  Fodor,  Nuttall,  Fliigge,  and 
others  demonstrate  that  the  defibrinated  blood  of  various 
vertebrates  destroys  anthrax-bacilli  in  test-tubes,  and  that 
this  peculiarity  of  the  blood,  however,  disappears  imme- 
diately on  exposure  to  a  temperature  of  55°  C.  (131°  F.). 
The  first  work  of  Behring  in  this  domain  tended  in  the  same 
direction.  Behring  made  the  discovery  that  the  blood  of  the 
white  rat  destroys  anthrax-bacilli,  and  he  made  therefrom 
the  deduction  that  the  immunity  of  the  rat  to  anthrax  is 
attributable  to  this  bactericidal  activity.  The  most  thor- 
ough investigations  into  these  relations  emanate  from  Buch- 
ner  and  his  pupils.  The  Munich  school  assumes  that  the 
blood-serum  and  the  bodily  fluids  derive  their  bactericidal 
property  from  special  substances — the  alexins — whose 
chemic  nature  has  not  yet  been  definitely  determined.  They 
are  precipitated  out  of  solution  by  alcohol,  are  destroyed 
by  exposure  for  from  half  an  hour  to  an  hour  to  tempera- 
tures of  from  55°  C.  (131°  F.)  to  60°  C.  (140°  F.),  and  are 
attenuated  by  brief  exposure  to  a  temperature  of  37°  C. 
(98.6°  F.),  and  by  prolonged  exposure  to  ordinary. tem- 
perature. In  the  absence  of  salts  the  alexins  are  absolutely 
innocuous.  For  this  reason  the  serum  loses  its  bactericidal 
activity  when  it  is  dialyzed,  or  when  it  is  diluted  with  eight 
or  ten  times  its  volume  of  distilled  water.  The  original 
bactericidal  activity  may,  however,  be  at  once  restored  by 
the  addition  of  sodium  chlorid  or  other  salts.  Ammonium 
sulphate  has  proved  most  active  in  this  connection,  increas- 
ing the  resistance  of  the  alexins  to  heat  quite  ten  degrees. 
The  destructive  influence  of  the  alexins  varies  with  relation 
to  individual  microorganisms.  It  may  be  well  developed 
toward  one  variety  of  bacteria,  and  entirely  wanting  with 
relation  to  another.  Between  these  two  extremes  there  are 
all  conceivable  gradations.  The  number  of  bacteria  ex- 
posed to  the  .action  of  the  bactericidal  serum  is,  however, 
always  of  considerable  importance,  as  even  the  most  active 
of  the^  bodily  fluids  are  incapable  of  destroying  more  than  a 
given  number  of  bacteria. 

In  spite  of  these  admittedly  correct  observations,  the  ex- 


IMMUNITY,    IMMUNIZATION,  AND   CURE.  61 

istence  of  immunity  is  not  to  be  explained  upon  the  basis 
of  this  bactericidal  activity,  as  there  is  a  want  of  the  neces- 
sary relation  between  the  degree  of  bactericidal  activity  and 
that  of  natural  immunity.  White  rats,  to  the  bactericidal 
activity  of  whose  blood  reference  has  already  been  made, 
are  not  absolutely  immune  to  anthrax,  and  the  blood-serum 
of  dogs,  which  possess  a  considerable  degree  of  immunity 
to  this  disease,  constitutes  an  admirable  culture-medium  for 
anthrax-bacilli.  It  is,  further,  questionable  whether  the 
alexins  exhibit  the  same  activity  in  the  organism  as  in  the 
test-tube.  According  to  Buchner's  view  this  is  the  case. 
Only  in  the  capillaries,  where  the  bacteria  accumulate  and 
are  thus  washed  by  but  little  blood,  is  the  influence  of  the 
alexins  not  equally  manifest. 

What  has  been  said  of  congenital  immunity  is  applicable 
also  to  acquired  immunity.  Only  in  exceptional  instances 
is  there  a  proper  relation  between  the  bactericidal  activity 
of  the  blood-serum  and  artificial  immunity — as,  for  instance, 
after  vaccination  of  guinea-pigs  against  the  vibrio  of 
Metschnikoff,  when  the  blood  acquires  bactericidal  activity. 

The  bactericidal  activity  of  the  remaining  bodily  fluids  is, 
in  general,  less  than  that  of  the  blood-serum.  Such  activity 
has  been  observed  in  the  aqueous  humor  of  the  eye,  in  all 
possible  exudates  and  transudates,  and  even  in  the  saliva 
and  nasal  mucus.  The  bactericidal  activity  of  all  these 
fluids  is,  however,  neither  constant  nor  proportionate  to  the 
degree  of  immunity  present. 

The  alexins  appear  to  arise  from  the  leukocytes,  and  to 
represent  their  secretory  products.  Denys  and  his  pupils 
demonstrated  experimentally  that  the  bactericidal  activity 
of  the  serum  increases  or  diminishes  with  the  larger  or 
smaller  number  of  leukocytes  respectively.  Buchner  and 
Hahn  obtained  analogous  results,  and  the  view  of  the 
Munich  school  may  be  expressed  as  follows  :  the  leuko- 
cytes constitute  an  important  factor  among  the  natural  pro- 
tective forces  of  the  organism  through  substances  in  solution 
secreted  by  them.  Metschnikoff  also  assumes  as  established 
the  fact  that  a  certain  relation  exists  between  the  bactericidal 
activity  of  the  blood  and  the  number  of  leukocytes  ;  and 
he  adds  that  with  the  death  of  the  phagocytes,  which  takes 
place  abundantly  on  abstraction  of  blood,  a  portion  of  these 
bactericidal  substances  is  set  free,  and  it  is  these  that  repre- 
sent a  large  portion  of  the  alexins  of  the  serum. 


62  CLINICAL  BACTERIOLOGY. 

2.  Lysogenic  Activity  of  the  Immune  Serum  (Pfeif- 
fer's  Reaction). — The  bactericidal  activity  of  the  bodily 
fluids  in  the  case  of  artificial  immunity  differs  from  that 
described  as  due  to  the  alexins,  as  was  discovered  by  R. 
Pfeiffer  and  his  pupils.  If  guinea-pigs  are  immunized  with 
carefully  destroyed  cultures  against  cholera-vibrios,  typhoid- 
bacilli,  coli  commune,  and  other  similar  microorganisms, 
the  animals  thus  treated  acquire  the  property  of  dissolving, 
after  intraperitoneal  introduction,  the  bacteria  toward  which 
they  have  been  immunized.  In  order  to  follow  this  phe- 
nomenon, which  is  now  generally  known  as  Pfeiffers  7'eac- 
tioti^  directly  under  the  microscope,  a  small  amount  of  the 
exudate  that  forms  in  the  abdominal  cavity  shortly  after  in- 
traabdominal injection  of  the  bacteria  is  removed,  from  time 
to  time,  by  means  of  capillary  glass  tubes.  Immediately 
after  the  injection  the  microscopic  field  exhibits  complete 
immobility  of  the  bacteria  ;  several  (up  to  ten)  minutes  later 
these  appear  swollen,  exhibit  beginning  disintegration  into 
granules,  and  after  the  lapse  of  ten  minutes  more  the  exu- 
date contains  only  fine  granules.  If,  at  this  time,  plates  are 
inoculated  with  the  fluid  obtained,  they  remain  sterile. 
Precisely  the  same  results  are  obtained  if,  instead  of  vacci- 
nated guinea-pigs,  animals  are  employed  that  were  not  pre- 
viously treated,  and  cholera-bacilli  or  typhoid-bacilli,  mixed 
with  a  minimal  amount  of  serum  from  an  animal  that  has 
been  immunized  against  cholera  or  typhoid  fever,  are  in- 
jected into  the  peritoneum.  In  this  experiment,  also,  dis- 
integration of  the  bacteria  into  fine  granules,  a  gradual  dis- 
solution within  the  infected  body,  is  observed. 

Pfeiffer  designates  as  bactericidal  such  serum  as  yields 
the  reaction  described  by  him.  This  designation,  however, 
has,  as  has  already  been  pointed  out,  been  employed  in 
another  sense — namely,  for  the  germ -destroying  action  of 
blood-serum  in  the  test-tube.  It  is,  therefore,  well  to 
adopt  the  suggestion  of  C.  Frankel,  who  designates  such 
serum  as  yields  the  phenomenon  of  Pfeiffer  as  lysogenic,  or 
solvent,  serum. 

Lysogenic  serum  tolerates  exposure  to  60°  C.  (140°  F.) 
for  an  hour,  its  bacterial  solvent  activity  being  thus  scarcely, 
if  at  all,  affected. 

Pfeiffer  himself  had  recognized  that  the  serum  of  normal 
animals  also  is  capable  of  inducing  the  reaction  described ; 
but  in  this  instance  it  is  necessary  to  inject  a  much  larger 


IMMUNITY,   IMMUNIZATION,  AND   CURE.  63 

amount  of  serum  than  when  the  serum  of  immunized 
animals  is  employed.  From  this  it  appears  that  the 
quantitative  relations  are  of  significance  with  regard  to  the 
reaction  of  Pfeiffer.  In  order  to  express  these  relations 
mathematically,  Pfeiffer  establishes  as  a  standard  or  a  unit 
for  the  serum  that  amount  that  is  just  necessary  when  in- 
jected simultaneously  into  the  peritoneal  cavity  to  destroy 
ten  times  the  minimal  lethal  amount  of  living  bacteria.  At 
least  0.05  cu.  cm.  (of  goat-serum,  however,  only  0.2 
cu.  cm.)  of  the  serum  of  a  normal  animal  are  necessary  for 
this  purpose  ;  whereas  only  one-tenth  of  a  milligram  of  the 
serum  of  a  highly  immunized  goat,  for  instance,  suffices. 
With  these  quantitative  limitations  the  reaction  of  Pfeiffer 
may  be  considered  specific.  Cholera-serum  manifests  its 
lysogenic  activity  in  the  peritoneal  cavity  of  guinea-pigs 
only  against  cholera-vibrios  ;  typhoid-serum  only  against 
typhoid-bacilli,  etc.  The  serum  of  individuals  who  have 
recovered  spontaneously  from  typhoid  fever  or  cholera  like- 
wise exhibits  the  phenomenon  of  Pfeiffer.  The  standard 
equals  about  0.0 1. 

The  reaction  of  Pfeiffer  may  be  admirably  employed  for 
purposes  of  differential  diagnosis.  With  its  aid  it  is  possi- 
ble to  differentiate  true  typhoid-bacilli  and  true  cholera- 
vibrios  from  the  great  horde  of  microorganisms  resembling 
typhoid-bacilli  and  cholera-bacilli  respectively.  It  need 
scarcely  be  emphasized  that  precisely  in  this  method  of 
differential  diagnosis  the  quantitative  relations  must  be 
most  carefully  considered. 

3.  Agglutination  (Gruber's  Reaction). — The  serum  of 
animals  immune  to  cholera,  typhoid  fever,  coli-infection, 
etc.,  or  of  human  beings  that  have  recovered  from  typhoid 
fever  or  cholera,  behaves  in  a  peculiar  manner  when  added 
in  small  amount  to  typhoid-bouillon,  cholera-bouillon,  coli- 
bouillon,  etc.  The  bacteria  lose  their  motility,  collect  in 
masses,  and  sink  to  the  bottom  of  the  test-tube  as  a  floccu- 
lent  precipitate  ;  whereas  the  supernatant  fluid  remains  per- 
fectly clear.  General  attention  was  directed  to  this  reaction 
of  immune  serum  through  the  labors  of  Gruber  and  Durham, 
and  somewhat  later  of  Pfeiffer  and  his  pupils,  although  a 
number  of  investigators,  and  more  particularly  Bordet,  had 
described  it  previously,  without,  however,  considering  it  of 
special  importance.  Gruber  introduced  the  term  aggluti- 
nation to  describe  the  phenomenon.     The  reaction  is  ob- 


64  CLINICAL  BACTERIOLOGY. 

tained  either  macroscopically  or  microscopically  in  one  of 
the  three  following  ways  : 

1 .  To  fresh,  sterile  bouillon,  in  carefully  measured  amount 
(p.  65),  is  added  one  drop  of  immune  serum,  and  the  mix- 
ture is  inoculated  with  cholera-bacilli,  typhoid-bacilli,  etc., 
and  then  placed  in  the  thermostat  at  37°  C.  (98.6°  F.). 
After  the  lapse  of  from  four  to  seven  hours  the  first  clumps 
appear  in  the  culture,  which  after  from  twelve  to  twenty- 
four  hours  presents  a  typical  appearance.  The  bacteria  are 
seen  deposited  at  the  bottom  of  the  test-tube  as  small  floc- 
culi,  in  a  certain  degree  precipitated,  while  the  overlying 
bouillon  is  perfectly  clear.  In  order  that  the  reaction  may 
not  be  overlooked,  it  is  advisable  to  inspect  the  fluid  quite 
frequently — if  possible,  from  hour  to  hour. 

2.  To  a  twenty-four-hour-old  bouillon-culture  immune 
serum  is  added,  as  in  the  previous  procedure,  and  the  mix- 
ture is  placed  in  the  thermostat  at  37°  C.  (98.6°  F.)  for 
from  one  to  eight  hours.  The  originally  turbid  culture 
soon  clears  up,  and  a  flocculent  precipitate  forms. 

3.  To  demonstrate  the  phenomenon  of  agglutination  mi- 
croscopically it  is  best  to  employ  young  bouillon-cultures 
not  more  than  twenty-four  hours  old.  In  the  employment 
of  older  cultures  there  is  danger  of  mistaking  for  actual 
agglutination  the  clump-formation  that  not  rarely  takes 
place  spontaneously,  especially  on  the  surface  of  the  fluid. 
The  observer  assures  himself  previously  through  control- 
preparations  that  the  bacilli  in  the  bouillon  employed  are 
actively  motile,  and  especially  that  they  are  distinctly  sepa- 
rated one  from  another.  Then  to  10,  30,  40,  100  (repre- 
senting 5  cu.  cm.),  200  (about  10  cu.  cm.),  1000  (about  50 
cu.  cm.)  or  even  a  larger  number  of  drops  of  this  bouillon  in 
sterile  Petri  dishes  is  added  one  drop  of  immune  serum, 
and  microscopic  preparations  are  made  of  the  various  mix- 
tures. If  the  serum  exhibit  agglutinating  activity,  a  num- 
ber of  confluent  masses  or  islands  of  bacteria  become  visible, 
as  a  rule.  In  these  the  microbes  are  absolutely  immobile, 
whereas  in  the  free  intervals,  a  greater  or  lesser  number  are 
at  first  still  in  active  movement.  Should  the  preparation 
exhibit  molecular  movement,  it  is  permitted  to  stand  for 
from  a  quarter  to  half  an  hour,  after  which  it  is  again  exam- 
ined. The  agglutination  is  favored  by  evaporation  and  by 
the  presence  of  ox;^gen.  To  prevent  evaporation,  the  ex- 
amination may  be  made  in  hanging  drop,  although  this  is 


IMMUNITY,   IMMUNIZATION,  AND   CURE.  65 

somewhat  inconvenient.  The  characteristic  islands  are  then 
seen  to  form  at  the  periphery  of  the  drop. 

Gruber's  reaction  can  also  be  obtained  with  the  serum  of 
normal  animals  and  human  beings,  but  in  this  instance  much 
more  serum  is  required  than  when  the  serum  of  immune 
animals  is  employed.  In  the  case  of  normal  serum  the  pro- 
portion in  the  large  majority  of  cases  exceeds  i  :  lo — that  is, 
the  addition  of  more  than  one  drop  of  serum  to  ten  drops 
of  bouillon  is  required  to  induce  agglutination.  Only  in 
rare  instances  has  a  proportion  of  i  :  30,  or  even  of  i  :  40, 
been  noted  in  human  beings.  The  serum  of  rabbits,  horses, 
asses,  possesses  agglutinating  activity  that  varies  between 
I  :  30  and  i  :  40.  The  serum  of  guinea-pigs,  as  a  rule,  ex- 
hibits no  agglutinating  property  whatever.  It  is,  therefore, 
absolutely  necessary  in  studying  the  agglutinating  reaction 
to  consider  the  quantitative  relations  in  every  instance.  In 
applying  the  test  according  to  either  the  first  or  the  second 
of  the  methods  described,  several  observations  are  always 
made.  To  the  bouillon  is  first  added  serum  in  the  propor- 
tion of  I  :  10,  in  a  second  test-tube  in  the  proportion  of  i  :  20, 
then  I  :  30,  i  :  50,  i  :  100,  etc. 

With  an  observance  of  the  precautionary  measures  noted 
the  reaction  of  Gruber  is  of  great  service  in  the  differential 
diagnosis  of  bacteria  that  resemble  one  another  morpho- 
logically. It  is  preferable  to  the  reaction  of  Pfeiffer,  as  it  is 
much  more  easily  performed.  To  identify  cholera-bacilli, 
typhoid-bacilli,  etc.,  the  serum  of  animals  immune  to 
cholera,  typhoid  fever,  etc.,  is  tested  to  determine  its  power 
of  causing  agglutination  of  bouillon-cultures  of  the  respect- 
ive organisms.  If  the  reaction  is  negative  when  a  dilution 
of  I  :  10  is  employed,  it  may  be  concluded  with  certainty 
that  the  bacteria  under  examination  are  not  identical  with 
those  to  which  the  animal  yielding  the  serum  has  been 
rendered  immune.  If,  however,  the  reaction  prove  posi- 
tive, the  result  is  in  favor  of  the  conclusion  that  the  micro- 
organisms are  the  same.  To  establish  completely  the 
identity  of  the  microorganism  it  is  necessary,  however,  to 
determine  how  far  the  degree  of  dilution  can  be  carried.  In 
individual  instances  this  may  reach  several  thousand.  The 
proportion  of  from  i  :  50  to  i  :  75  is,  however,  quite  suffi- 
cient, and  such  a  dilution  will  be  satisfactory  if  but  a  small 
amount  of  serum  is  available.  The  extreme  limit  of  agglu- 
tinating activity  is  best  determined  with  the  aid  of  the 
5 


66  CLINICAL  BACTERIOLOGY. 

microscope.  When  excessive  dilutions  are  employed,  the 
reaction  may  escape  macroscopic  observation ;  whereas, 
under  the  microscope,  after  a  time  (from  one  minute  to  two 
hours),  agglutination  may  yet  be  observed. 

In  addition  to  typhoid-cultures,  cholera-cultures,  and 
coli-cultures,  the  reaction  of  Gruber  has  been  demonstrated 
with  numerous  other,  and  also  nonpathogenic,  bacteria. 
The  agglutinating  property  of  the  serum  is,  further,  not 
present  immediately  after  introduction  of  the  bacteria  into 
the  animal  body ;  at  least  three  and  a  half,  and  generally 
even  five,  days  must  elapse  before  this  appears.  Besides  the 
blood-serum,  the  reaction  of  Gruber  may  be  obtained  in  an 
intense  degree  with  the  serous  contents  of  blisters  induced 
by  vesication,  in  lesser  degree  with  milk,  and  in  still  less 
degree  with  urine,  dropsical  fluid,  exudates,  bile,  tears,  and 
aqueous  humor  of  the  respective  animals  and  human  beings. 

Gruber  considered  the  phenomenon  of  agglutination  as 
a  reaction  of  immunity,  and  attempted  to  base  upon  it  a  new 
theory  of  immunity,  to  which  we  shall  later  refer.  A  not 
inconsiderable  advance  was  made  in  this  connection  when 
Widal  showed  that  agglutination,  at  least  in  human  beings, 
represents  a  reaction  of  the  period  of  infection.  Widal  dem- 
onstrated that  the  serum  of  typhoid-fever  patients  at  the 
end  of  the  first  or  at  the  beginning  of  the  second  week  of 
the  disease  most  distinctly  yields  the  agglutination-phe- 
nomenon with  typhoid-bacilli  (Widal-Gruber  reaction). 
This  fact  is  of  the  greatest  importance  from  a  practical 
point  of  view.  It  renders  possible  the  diagnosis  of  typhoid 
fever  with  the  aid  of  the  serum  of  a  suspected  patient.  The 
same  appears  to  be  true  for  Asiatic  cholera  and  other  dis- 
eases. We  shall  fully  discuss  the  so-called  serum-diagnosis 
in  the  special  section.      (See  Typhoid  Fever.) 

The  nature  of  the  agglutinating  substance  has  not  yet 
been  determined  despite  numerous  investigations,  especially 
on  the  part  of  Widal.  It  appears  to  be  quite  resistant,  as 
exposure  of  the  serum  to  a  temperature  of  60°  C.  (140°  F.) 
for  an  hour  fails  to  abolish  the  agglutinating  phenomenon  ; 
this  result  is  obtained  only  after  exposure  to  a  temperature 
of  80°  C.  (176°  F.).  The  reaction  of  Gruber  may  be 
obtained  also  with  dead  bacilli.  The  bacteria  are  best  de- 
stroyed with  formol,  and  they  can  then  be  kept  for  weeks 
without  losing  in  the  slightest  degree  their  sensitiveness  to 
the  action  of  the  serum.      From  this  Widal  arrives  at  the 


IMMUNITY,    IMMUNIZATION,  AND   CURE.  67 

conclusion  that  the  agglutination  is  not  a  manifestation  of 
the  vital  activity  of  the  bacilli,  but  rather  that  it  represents 
a  passive  reaction  on  the  part  of  the  protoplasmic  sub- 
stance. According  to  a  more  recent  statement  by  Kraus, 
a  mixture  of  immune  serum  with  filtered  cultures,  after 
exposure  for  twenty-four  hours  to  a  temperature  of  37°  C. 
(98.6°  F.),  exhibits  a  precipitate,  and  sometimes  even  the 
formation  of  flocculi.  According  to  Gruber,  the  agglutinat- 
ing substances  are  derived  from  the  bodily  constituents  of  the 
bacteria.  This  statement,  however,  can  not  be  sustained, 
as  agglutinating  properties  may  be  observed  in  the  serum 
of  animals  after  the  injection  of  soluble  metabolic  products, 
of  filtered,  quite  young  bouillon-cultures. 

We  have  already  stated  that,  as  the  result  of  his  investi- 
gations of  agglutination,  Gruber  attempted  to  establish  a 
new  theory  of  immunity.  This  assumes  that  the  aggluti- 
nating substances  cause  the  bacterial  membrane  to  swell, 
and  thus  render  the  bacterial  protoplasm  accessible  to  the 
alexins  of  Buchner,  in  consequence  of  which  death  of  the 
microorganisms  is  brought  about.  This  hypothesis  of 
Gruber,  however,  is  not  supported  by  the  facts  elicited  by 
further  investigation.  There  need  be  no  relation  between 
agglutination  and  immunity.  There  are  cases  in  which,  in 
spite  of  the  agglutinating  property  of  the  serum,  immunity 
does  not  exist,  and  vice  versa.  Further,  the  assumed  swell- 
ing of  the  bacilli  can  not  be  demonstrated  microscopically. 

4.  Antitoxins. — The  anti-substances  of  the  bodily  fluids, 
and  especially  of  the  blood-serum,  thus  far  described,  mani- 
fest their  activity,  on  the  whole,  directly  against  the  bacteria 
themselves.  The  conditions  are  quite  otherwise  with  that 
class  of  anti-substances  that  are  the  last  to  be  discussed,  and 
are,  probably,  the  most  important.  We  have  reference  to 
the  substances  designated  antitoxins,  which  derive  their 
name  from  the  fact  that  their  energy  is  directed  not  so 
much  against  the  microorganisms,  but  rather  against  the 
metabolic  products  (toxins)  generated  by  them.  Their  dis- 
covery in  the  case  of  diphtheria  and  of  tetanus  is  the  funda- 
mental work  of  Behring,  and  the  stimulus  to  their  further 
investigation  was  given  especially  by  Ehrlich.  The  latter 
found  in  ricin  and  in  abrin — two  albuminoid  vegetable 
poisons — substances  presenting  numerous  points  of  resem- 
blance to  bacterial  poisons,  and  with  which  some  of  the  laws 
of  immunity  can  be  readily  studied.     As  a  result  of  his 


68  CLINICAL  BACTERIOLOGY. 

experiments  with  these  substances,  EhrHch  recognized  that 
in  the  process  of  immunization  through  the  activity  of  the 
disease-poisons  in  the  body  of  the  patient,  substances  are 
formed  that  he  designates  anti-bodies  (antitoxins).  These 
are,  in  a  certain  sense,  antidotes,  inasmuch  as  they  neutral- 
ize or  prevent  the  toxic  action  of  the  disease-poisons. 
When  these  antitoxins  are  present  in  sufficient  amount,  im- 
munity exists.  In  the  process  of  immunization  with  the  aid 
of  bacteria  or  their  poisons  {direct,  active,  immediate  immu- 
nizatioji),  the  antitoxins  are  formed  either  from  the  bac- 
terial products  themselves,  or,  under  their  influence,  from 
substances  that  exist  in  the  body  preformed. 

The  disease  continues  until  an  adequate  amount  of  anti- 
toxin has  been  formed.  For  this  reason  the  older  methods 
of  immunization  are  effective  only  after  disease  of  greater  or 
less  severity,  and  after  the  lapse  of  a  certain  time.  Immu- 
nization by  means  of  serum  (^passive,  mediate,  indirect  im- 
munization) represents  the  transmission  of  preformed  anti- 
toxins. Therefore,  this  method,  on  the  one  hand,  induces 
no  disease,  and,  on  the  other  hand,  it  establishes  immunity 
at  once  ;  and  this  is,  for  the  same  reason,  also,  more  transi- 
tory, lasting  only  a  few  weeks. 

Whence  the  antitoxins  are  derived  has  not  yet  been 
finally  determined.  The  view  that  the  antitoxin  is  formed 
directly  from  the  toxin  introduced  for  purposes  of  immuni- 
zation appears  gradually  to  be  losing  ground.  It  is  more 
probable  that  in  the  course  of  every  toxic  disease  the  anti- 
toxin is  produced  together  with  the  toxin  within  the  body. 
The  antitoxic  activity  of  the  serum  is  abolished  by  exposure 
to  a  temperature  of  from  60°  (140°  F.)  to  70°  C.  (158°  F.). 

The  manner  in  which  antitoxins  act  upon  the  bacterial 
poisons  has  not  yet  been  clearly  determined.  Originally, 
it  was  assumed  that  the  antitoxins  destroy  the  bacterial  poi- 
sons. The  injection  of  a  mixture  of  an  antitoxin-containing 
serum  and  bacterial  poison  proved  innocuous.  From  this 
it  was  concluded  that  the  poison  is  destroyed  by  the  anti- 
toxin of  the  serum.  It  soon  transpired,  however,  that  de- 
struction of  the  poison  by  the  antitoxin  does  not  take  place, 
but  that,  to  use  the  expression  of  Ehrlich,  in  the  physio- 
logically neutral  mixtures  of  toxin  and  antitoxin  both  sets 
of  constituents  are  yet  present  as  such.  Buchner  and 
Roux  assumed  an  action  of  the  antitoxin  upon  the  cells,  as 
a  result  of  which  the  latter  are  rendered  immune  to  the  in- 


IMMUNITY,   IMMUNIZATION,   AND   CURE.  69 

toxication.  In  contradistinction  from  this  cellular  hypothe- 
sis is  the  chemic  view  of  Behring  and  Ehrlich  :  that  toxin 
and  antitoxin  undergo  a  sort  of  double  combination,  which 
proves  innocuous  for  the  tissues.  The  decision  of  this  ques- 
tion appears  finally  to  have  been  made  in  favor  of  the 
chemic  theory  by  the  more  recent  investigations  of  Ehrlich 
with  regard  to  ricin  and  antiricin.  Ricin  has  the  property 
of  causing  the  red  blood-corpuscles  in  defibrinated  blood  to 
collect  together  and  to  be  precipitated  to  the  bottom  of  the 
vessel — a  process  from  which  vital  processes  may  with  cer- 
tainty be  excluded.  Ehrlich  showed,  then,  that  antiricin, 
which  is  present  in  the  blood-serum  of  animals  immunized 
to  ricin,  abolishes  the  activity  of  ricin  in  the  test-tube,  and 
that  the  peculiar  coagulation  following  the  addition  of  ricin- 
serum  no  longer  takes  place.  Ricin  and  antiricin  must,  in 
this  instance,  have  directly  influenced  each  other  chemic- 
ally. Ehrlich  was  further  able  to  demonstrate  that  the 
combination  of  toxin  and  antitoxin  takes  place  much  more 
quickly  in  concentrated  than  in  dilute  solutions,  that  heat 
hastens  and  cold  retards  its  occurrence.  As  similar  mani- 
festations are  observed  in  chemistry  in  the  formation  of 
double  salts,  it  would  seem  probable,  according  to  Ehrlich, 
that  also  the  neutralization  of  toxins  by  antitoxins  repre- 
sents the  formation  of  a  double  salt. 

The  immunization  of  mammals  against  toxins  is  always 
attended  with  febrile  reaction,  and  it,  therefore,  appeared 
that  the  formation  of  an  antitoxin  would  not  be  possible  in 
the  absence  of  fever.  Metschnikoff,  however,  found  that 
of  all  animals  the  crocodile  produces  antitoxin  most  abund- 
antly and  most  speedily,  in  spite  of  the  fact  that  febrile 
movement  does  not  take  place. 

According  to  the  investigations  of  Behring  and  his  col- 
laborators, the  antitoxin  distributes  itself  throughout  the 
organism  in  man  and  in  animals  in  such  a  manner  that  after 
absorption  of  the  injected  serum,  after  passive  immuniza- 
tion, the  blood  to  a  certain  extent  extracts  the  antitoxin 
from  the  tissues  and  stores  it  up.  Twenty-four  hours  after 
subcutaneous  injection  of  serum,  and  in  a  shorter  time  after 
intravenous  or  intraperitoneal  injection,  the  maximum 
amount  of  antitoxin  in  the  blood  is  demonstrable.  The 
antitoxin  is  absorbed  from  stomach  and  bowel  only  when 
lesions  of  the  mucous  membrane  exist.  The  maximum 
content  of  antitoxin  in  the  blood  persists  for  several  days. 


70  CLINICAL  BACTERIOLOGY. 

After  this,  the  amount  of  antitoxin  in  the  blood  gradually 
diminishes.  It  now  appears  in  the  milk,  in  the  urine,  etc., 
until  finally  it  is  wholly  swept  out  of  the  body.  The 
rapidity  with  which  this  elimination  of  antitoxin  takes  place 
is  most  variable,  in  accordance  with  the  different  conditions 
present.  It  is  the  greater  the  larger  the  amount  of  immu- 
nizing serum  injected.  In  the  case  of  diphtheria,  the  pro- 
tection in  human  beings  following  the  usual  immunization 
with  250  antitoxin  normal  units  lasts  about  four  weeks. 

Success  in  the  preparation  of  antitoxic  serum  may  be 
hoped  for  only  when  the  poison,  the  toxin  of  the  respective 
species  of  bacteria,  is  known,  and  can  be  prepared  of  suffi- 
cient strength.  It  has  thus  far  been  possible  to  obtain  a 
high  degree  of  toxin-immunity  only  in  the  case  of  diph- 
theria, tetanus,  botulism,  snake-venom,  ricin,  and  abrin. 

It  has  already  been  mentioned  that  the  lysogenic  and  the 
agglutinating  substances  of  the  serum  are  also  present  in 
the  blood  of  normal  individuals.  The  same  statement 
applies  also  to  the  antitoxins.  Attention  has  been  called 
by  a  number  of  observers  to  a  certain  neutralizing  activity 
on  the  part  of  the  normal  blood-serum  of  horses  and  of 
human  beings  against  the  poison  of  diphtheria  and  other 
similar  poisons.  The  natural  antitoxic  pozver  of  the  blood- 
serum  is,  however,  only  slight.  It  by  no  means  attains  the 
high  degree  of  activity  exhibited  by  the  serum  of  animals 
artificially  made  poison-proof 

From  the  fact  that  immunity  can  be  transmitted  by 
means  of  serum  containing  antitoxin,  the  doctrine  that 
protection  against  toxins  is  the  cause  of  immunity  has  been 
brought  forward.  Behring,  and  also  Ehrlich,  ascribe  the 
real  cause  of  acquired  immunity  to  the  antitoxic  property 
of  the  blood.  However  attractive  this  theory  may  be,  all 
of  the  existing  facts  can  not  be  brought  in  harmony  with 
it.  Reference  may  be  made  to  the  instances  of  a  want  of 
relation  between  the  occurrence  of  antitoxin  in  the  blood 
(immunizing  capability  of  the  blood-serum)  and  the  pres- 
ence of  immunity  (p.  55).  It  was  mentioned  that  the 
occurrence  of  antitoxin  in  the  blood  of  animals  by  no 
means  establishes  necessarily  a  condition  of  immunity  in 
the  latter,  and  that,  under  certain  circumstances,  the  or- 
ganism whose  blood-serum  possesses  pronounced  immu- 
nizing capability,  exhibits  not  only  not  an  increased,  but 
even  a  diminished,  degree   of   resistance  to    the   bacterial 


IMMUNITY,    IMMUNIZATION,    AND   CURE.  71 

poisons  (Behring's  hypersensibility)  ;  while,  on  the  other 
hand,  marked  immunity  may  exist  without  the  presence  of 
antitoxins  in  the  blood. 

As  the  result  of  such  observations,  a  distinction  has 
been  made  between  tissue -immunity  and  serum-immunity 
{antitoxin-immunity\  The  latter  is  a  transitory  condition, 
dependent  upon  alterations  in  the  blood-mixture  from  the 
presence  of  the  circulating  toxins  ;  the  first  is  a  permanent 
condition,  dependent  upon  changes  in  the  tissues,  upon  the 
activity  of  the  cells,  which  have  become  insusceptible  to 
the  poisons.  Tissue-immunity,  or  histogenic  immunity,  is 
not  to  be  referred  to  the  presence  of  antitoxins.  Fowl, 
which  are  highly  immune  to  tetanus,  possess  little  if  any 
antitoxin  ;  but  their  blood  becomes  at  once  antitoxic  after 
injection  of  tetanus-toxin. 

Recently,  Behring  has  returned  to  his  original  view,  and 
believes  that  acquired  toxin-immunity,  active  as  well  as 
passive,  is  always  hematogenous — that  is,  dependent  upon 
the  antitoxic  activity  of  the  serum.  As  histogenic  he  con- 
siders only  the  natural  immunity  to  the  bacterial  poisons. 
Finally,  the  antitoxins  alone  are  as  incapable  as  the  other 
anti-bodies  or  as  phagocytosis  of  explaining  all  of  the 
manifestations  of  immunity. 

We  have  in  the  foregoing  presented,  as  objectively  as 
possible,  the  facts  that  investigation  in  the  domain  of 
immunity  have  developed  in  great  abundance.  They  do 
not  permit  of  the  establishment  of  a  single,  universally 
applicable  theoiy  of  immunity.  They  rather  render  it 
probable  that  there  is  no  such  unity,  but  that  immunity  is 
eventually  not  a  simple  and  indivisible  process,  dependent 
in  all  cases  upon  one  and  the  same  basis,  but,  apparently, 
variable  and  complex  in  its  nature,  dependent  in  one  in- 
stance upon  this,  in  another  instance  upon  that,  cause,  and 
more  frequently  due  to  several  in  combination. 

Relations  Between  Immunity  and  Cure. — In  the  case 
of  scarlet  fever,  measles,  and  other  like  diseases,  recovery 
from  an  attack  is  attended  with  immunity.  If  in  the  case 
of  other  diseases — as,  for  instance,  pneumonia,  erysipelas, 
etc. — recovery  from  one  attack  rather  predisposes  to  subse- 
quent attack,  this  does  not  exclude  the  fact  that  at  the  time 
of  recovery  immunity  existed,  a  so-called  temporary  im- 
munity, which  disappears  in  the  course  of  a  few  days  or 
weeks.     It  is  noteworthy  in  this  connection  that  the  dem- 


72  CLINICAL  BACTERIOLOGY. 

onstration  was  first  made  experimentally  for  pneumonia, 
later  for  typhoid  fever,  diphtheria,  and  cholera,  that  the 
blood  of  individuals  convalescent  from  these  diseases  ex- 
hibits in  many  instances  transitory  immunizing  activities 
with  relation  to  the  respective  bacteria.  It  appears  from 
this  as  if  recovery  from  these  diseases  also  is  attended  with 
immunity,  but  that  this — through  causes  not  yet  made 
clear — is  a  quickly  passing  one. 

It  has  been  demonstrated  experimentally  with  certainty 
that  immunization  may  also  lead  to  cure.  By  means  of 
serum-immunization  it  is  possible,  if  the  serum  is  derived 
from  animals  highly  enough  immunized,  to  induce  curative 
results,  even  when  the  treatment  is  begun  a  certain  time 
after  infection  has  taken  place.  These  facts  are  most  con- 
clusively demonstrable  experimentally  in  the  case  of  tetanus. 
With  large  amounts  of  serum  from  animals  immunized  to 
tetanus,  it  is  possible  to  save  mice  and  guinea-pigs  that 
already  exhibit  distinct  tetanic  manifestations.  We  shall 
refer  more  fully  to  these  relations  in  the  special  section. 
(See  Diphtheria  and  Tetanus.) 

According'^to  the  foregoing,  two  facts  have  been  demon- 
strated— in  the  first  place,  that  immunity  exists  at  the  time 
of  recovery  in  the  sequence  of  toxic  diseases  in  man,  and,  in 
the  second  place,  that  it  is  possible  experimentally  to  cure 
infection  by  the  timely  establishment  of  immunity.  From 
this  the  conclusion  may,  with  all  probability,  be  drawn  that 
also  in  human  beings  the  connection  between  recovery  and 
immunity  consists  in  the  bringing  about  of  recovery  through 
the  development  of  immunity ;  recovery  from  the  given 
disease  immunizes  the  organism,  and  cure  is  effected  in 
consequence  of  immunization  naturally  induced,  and  espe- 
cially the  crisis  appears  as  the  expression  of  cure  through 
the  sudden  setting  in  of  immunity.  Upon  this  knowledge 
is  based  recent  therapeutic  effort,  which  is  known  as 
serum-therapy  or  immunization-therapy.  The  object  to  be 
attained  is  the  immunization  of  the  diseased  organism  after 
infection  has  taken  place — that  is,  the  effecting  of  a  cure  in 
the  same  way  as  nature  brings  about  recovery  in  cases  of 
infectious  disease  pursuing  a  favorable  course.  This  mode 
of  therapy  is  naturally  specific,  as  immunity  also  is  specific. 
Much  more  serum,  however,  is  required  to  effect  a  cure 
than  to  induce  immunity ;  or,  what  amounts  to  the  same 
thing,  the  serum  of  much  more  highly  immunized  animals. 


IMMUNITY,    IMMUNIZATION,   AND   CURE.  73 

Recent  experiments  of  Donitz  with  tetanus-antitoxin 
show,  further,  in  a  most  conclusive  manner,  that  the  amount 
of  serum  necessary  for  curative  purposes  is  the  greater  the 
longer  the  period  of  time  that  has  elapsed  between  the  in- 
toxication and  the  institution  of  serum-therapy.  Eight 
minutes  after  tetanus-intoxication,  according  to  Donitz,  six 
times  as  much  serum  is  required  in  order  to  save  the 
animal  as  when  the  serum  is  injected  immediately  after  the 
poison  ;  after  an  hour  the  curative  dose  is  twenty-four 
times  the  original  dose ;  and  so  on,  until  finally  a  period 
is  reached  at  which  it  is  entirely  impossible  to  save  the 
animal,  even  with  the  largest  amount  of  the  most  active 
serum.  For  this  reason  it  is,  above  all  things,  essential  in 
obtaining  serum  for  therapeutic  purposes  to  establish  in 
the  animals  yielding  the  blood  (generally  horses)  as  high 
a  degree  of  immunity  as  possible.  The  higher  the  degree 
of  immunization  established,  the  smaller  the  amount  of 
serum  required  to  effect  cure.  In  making  the  degree  of 
immunity  as  high  as  possible  it  must  be  borne  in  mind  that 
the  immunizing  process  pursues  a  wave-like  course  (Brieger 
and  Ehrlich).  Immediately  after  introduction  of  the  next 
higher  toxic  dose  the  immunizing  power  of  the  blood-serum 
diminishes.  It  remains  for  a  few  days  at  the  lower  level, 
gradually  rising  again  until  it  reaches  the  maximum.  From 
this  point  it  sinks  again,  and  it  finally  reaches  a  level  at 
which  it  persists  for  weeks.  The  most  favorable  time  for 
injecting  new  toxin  into  the  animals  in  order  further  to 
fortify  their  immunity  is  when  the  strength  of  the  serum  is 
highest — that  is,  when  the  anti-bodies  are  present  in  the 
body  in  largest  number. 

The  efforts  in  the  domain  of  curative  serum-therapy  have 
already  yielded  material  practical  results  in  the  case  of 
diphtheria.  In  that  of  tetanus  success  is  as  yet  doubtful. 
We  shall  refer  fully  in  the  special  section  in  the  discussion 
of  these  two  diseases  to  the  mode  of  obtaining  and  of  esti- 
mating and  to  the  dosage  of  the  curative  serum. 

Naturally,  immunization  is  not  the  only  manner  in  which 
therapeutic  attack  upon  bacterial  diseases  is  to  be  made. 
An  infectious  disease  may  be  terminated  by  reason  of  the 
death  of  the  bacteria  in  the  body.  It  would  be  possible  to 
effect  cure  in  this  way  if  the  infectious  agent  could  be  de- 
stroyed within  the  body  by  means  of  internal  disinfection. 
In  spite  of  the  large  number  of  antiseptics  at  our  command, 


74  CLINICAL  BACTERIOLOGY. 

and  in  spite  of  their  promptness  in  destroying  bacteria  in 
test-tubes,  they  are  not  appHcable  to  the  Hving  body,  be- 
cause they  either  fail  or,  employed  in  the  necessary  strength, 
destroy  not  only  the  bacteria,  but  also  the  cells  of  the  body. 
The  possibility,  however,  that  a  disinfectant  may  yet  be 
found  that  will  destroy  only  the  bacteria  without  affecting 
the  tissues,  must  remain  an  open  one. 


IV*    METHODS  OF  CULTURE  AND  OF  EXAMINA- 

TION- 

STERILIZATION. 

In  order  to  follow  the  individual  varieties  of  bacteria 
perfectly  in  the  course  of  their  development,  and  in  order 
to  employ  them  in  animal  experimentation,  ij;  i$  necessary 
to  obtain  them  in  pure  adttirc.  "  In  the  making  of  such 
pure  cultures  the  most  scrupulous  care  must  be  observed 
to  exclude  the  large  number  of  bacteria  that  are  every- 
where present.  The  instruments  employed  in  the  various 
manipulations,  the  nutrient  media,  and  the  vessels  that 
serve  as  the  field  of  development  for  the  bacteria,  must  be 
absolutely  germ-free — sterile.  To  accomplish  sterilization 
of  these,  ordinary  antiseptic  measures  can  not  be  employed, 
as  the  addition  of  substances  capable  of  destroying  the 
germs  or  of  inhibiting  their  activity  would  naturally  render 
the  nutrient  media  unsuitable  for  culture-purposes.  For 
this  reason  heat  exclusively  is  employed  for  the  sterilization 
of  all  materials  used  in  the  culture  of  bacteria,  and  both 
dry  heat,  as  well  as  moist  heat,  in  the  form-  of  live  steam. 

Dry  Heat. — As  dry  heat  penetrates  but  slowly  into  the 
interior  of  objects,  it  is  employed  principally  for  the  steril- 
ization of  articles  of  small  volume  only ;  thus,  platinum- 
needles  are  sterilized  directly  by  exposure  to  the  flame  of  a 
spirit-lamp  or  of  a  Bunsen  burner,  and  other  instruments 
by  being  moved  to  and  fro  for  about  a  minute  immediately 
above  the  flame.  Articles  made  of  glass  and  other  sub- 
stances that  tolerate  high  temperatures  are  placed  in  a 
double-walled  sheet-iron  receptacle  covered  with  asbestos 
(drying  chamber),  which  is  heated  to  between  150°  C. 
(302°  F.)  and  ;70°  C.  (338°  F.)  by  means  of  a  gas-flame 
burning  beneath  it.  (Fig.  9.)  After  exposure  for  half  an  hour 
to  air  thus  heated  to  from  150°  C.  (302°  F.)  to  170°  C. 


METHODS  OF   CULTURE  AND  OF  EXAMINATION. 


75 


(338°  F.),  even  the  most  resistant  spores  are  destroyed.  It 
suffices,  further,  in  this  mode  of  sterihzation,  to  heat  the 
drying  chamber  in  which  the  instruments  to  be  steriHzed  are 
placed,  until  a  thermometer  introduced  from  the  top  indi- 
cates a  temperature  of  170°  C.  (338°  F.) ;  then  the  supply 
of  gas  is  cut  off,  and  after  the  apparatus  has  completely 
cooled,  the  now  sterile  contents  are  removed. 


Fig.  9.— Hot-air  sterilizer. 


Live  Steam. — Most  substances,  however,  that  are  em- 
ployed in  bacteriologic  investigations,  especially  the  nutri- 
ent media,  do  not  bear  sterilization  by  means  of  such  high 
degrees  of  heat  as  have  been  mentioned ;  they  are,  there- 
fore, rendered  germ-free  by  means  of  live  steam.  For  this 
purpose  they  are  introduced  into  a  cylindric  apparatus, 
made  of  galvanized  iron  or  of  copper,  and  covered  with  felt 
or  with  asbestos.  (Koch's  steam-chamber.  Figs.  10,  ii.) 
This  vessel  is  divided  by  means  of  a  perforated  shelf  or  a 
wire  grating   into   an    upper   larger  and  a  lower   smaller 


76 


CLINICAL   BACTERIOLOGY. 


space.  The  former  is  intended  to  receive  the  substances  to 
be  steriHzed,  while  the  latter  is  intended  for  the  water.  The 
bottom  of  the  cylinder  is  heated,  the  water  is  made  to  boil, 
and  the  steam  generated  streams  through  the  perforated 
partition  into  the  upper  sterilizing  chamber,  which  is  closed 
by  means  of  a  loosely  applied  cover.  Exposure  to  live 
steam  for  from  half  an  hour  to  an  hour,  according  to  the 


Fig.  10. — Koch's  steam  sterilizer. 


Fig.  II. — Koch's  steam-chest. 


amount  of  fluid  or  the  size  of  the  articles  to  be  sterilized, 
suffices,  as  a  rule,  to  free  these  from  germs. 

It  is  useful,  when  an  adequate  water-supply  is  available, 
to  connect  the  Koch  steam-cylinder  with  a  constant  water- 
bath.  For  sterilization  with  steam  under  pressure  at  a  tem- 
perature of  I  io°  C.  (230°  F.)  (almost  i  i^  atmospheres)  or 
120°  C.  (248°  F.)  (two  atmospheres  of  pressure),  special 
apparatus  (digesters,  autoclaves)  are  required,  whose  con- 


METHODS  OF  CULTURE  AND  OF  EXAMINATION.         77 

struction  is  rather  expensive.  Disinfection  under  increased 
pressure  has  the  great  advantage  that  it  is  effected  much 
more  rapidly;  with  a  temperature  of  120°  C.  (248°  F.), 
about  fifteen  or  twenty  minutes  are  required  to  destroy  all 
germs,  even  the  especially  resistant  spores  of  some  bacilli  of 
food  and  of  earth  which  are  not  destroyed  with  certainty  by 
free  steam  even  after  an  exposure  of  five  hours.  The  objection 
has  been  raised  to  disinfection  with  steam  under  pressure 
that  it  does  not  sterilize  with  certainty  because  a  tempera- 
ture of  120°  C.  (248°  F.)  does  not  prevail  in  all  parts  of  the 
autoclave.  This  objection  is,  however,  not  justifiable.  It 
is  only  necessary  to  be  certain  that  no  trace  of  air  is  left 
in  the  apparatus,  and  to  this  end  the  valve  is  closed  after 
generation  of  steam  has  been  taking  place  for  five  minutes. 
The  sterilization  of  certain  fluids  containing  albuminoid 
substances  requires  special  precautions.  Live,  or  even 
compressed,  steam  can  not  be  employed  for  this  purpose, 
inasmuch  as  coagulation  would  take  place.  Resort  must, 
therefore,  be  had  to  so-called  fractional,  or  discontinuous, 
sterilization  (Tyndall).  The  fluids  to  be  disinfected  are  ex- 
posed for  four  or  five  hours  to  a  constant  temperature  of 
from  56°  C.  (132.8°  F.)  to  58°  C.  (136.4°  F.).  Exposure 
for  four  hours  to  a  temperature  of  58°  C.  (136.4°  F.)  is 
sufficient  to  destroy  most  developed  bacteria.  The  spores 
that  remain  in  consequence  of  their  greater  resistance  are 
now  permitted  to  germinate,  by  leaving  the  fluid  undisturbed 
for  twenty-four  hours.  After  the  lapse  of  this  interval,  the 
fluid  is  again  exposed  for  four  hours  to  a  temperature  of 
from  56°  C.  (132.8°  F.)  to  58°  C.  (136.4°  F.) ;  and  this 
mode  of  procedure  is  repeated  daily  for  a  whole  week.  At 
the  end  of  this  time  all  of  the  spores  will  have  developed 
into  bacteria,  and  these  will  in  turn  have  been  destroyed. 
This  method  is  not,  however,  trustworthy  under  all  condi- 
tions, as  spores  may  develop  after  the  lapse  of  a  week.  It 
appears  better  to  introduce  the  albuminoid  fluids  (blood- 
serum,  etc.)  in  small  amounts  into  test-tubes  or  the  like 
(Fig.  1 2),  and  to  expose  these  for  a  considerable  length  of 
time  (from  four  to  six  hours)  to  a  temperature  of  from 
56°  C  (132.8°  F.)  to  58°  C.  (136.4°  F.),and  then  to  place 
them  for  two  days  in  the  thermostat  at  a  temperature  of 
37°  C.  (98.6°  F.).  The  tubes  in  which  contaminations 
appear  are  set  aside.  With  care  in  manipulation  the  number 
of  turbid  tubes  will  rarely  be  large.     Fractional  sterilization 


78 


CLINICAL  BACTERIOLOGY. 


naturally  affords  no  protection  against  the  thermophilic  bac- 
teria (p.  22).  After  sterilization  has  been  effected,  the  ves- 
sels and  nutritive  media  must,  as  a  matter  of  course,  be 
protected  against  all  subsequent  contamination,  especially 
through  atmospheric  germs.  For  this  purpose  the  orifices 
of  the  tubes  are,  even  before  they  are  filled,  closed  by  means 
of  cotton  stoppers  ;  the  tubes  are  then  sterilized  by  exposure 
to  dry  heat  at  a  temperature  of  170°  C.  (338°  F.),  and  the 
fluid  is  introduced  into  them.  The  cotton  filters  the  air, 
and  restrains  the  entrance  of  the  germs.  If  molds  find  their 
way  upon  the  cotton  stopper,  they  may,  under  certain  cir- 
cumstances, if  the  tubes  have  been  kept  for  a  long  time, 
penetrate  the  cotton  with  their  mycelial  filaments.  This 
undesirable  occurrence  is  to  be  avoided  by  cutting  off  the 
excess  of  cotton,  exposing  the  free  surface  in  the  flame,  and 


Fig.  12. — I,  A  tube  of  blood-serum  ;  2,  a  sterilized  cotton  swab  in  test-tube. 


then  applying  over  it  a  closely  fitting  rubber  cap  that  has 
been  previously  disinfected  in  a  I  :  looo  solution  of  mercuric 
chlorid. 

Preparation  of  Nutrient  Media. — Whereas,  previously 
to  the  time  of  Robert  Koch,  we  were  restricted  almost  ex- 
clusively to  the  employment  of  fluid  nutrient  media,  Koch, 
through  the  addition  of  gelatinous  substances — that  is,  such 
as  become  fluid  when  heated,  and  solid  again  when  subse- 
quently cooled — introduced  the  use  of  solid,  and  at  the 
same  time  transparent,  culture-media,  which  made  it  possi- 
ble to  isolate  individual  colonies  of  bacteria  and  to  study 
their  development. 

The  nutrient  media  that  are  most  generally  employed  for 
the  cultivation  of  pathogenic  bacteria  are  bouillon,  gelatin, 
agar-agar,  blood-serum,  and  potato. 

(a)  Preparation  of  Bouillon  (Loffler). — One  pound  of 
chopped  meat  freed  from  fat  and  tendon  is  macerated  for 


METHODS   OF  CULTURE  AND  OF  EXAMINATION.        79 

from  twelve  to  twenty-four  hours  in  a  liter  of  water  (at 
summer-temperature  in  a  refrigerator).  The  infusion  is 
squeezed  through  a  clean  cloth  by  means  of  a  press,  or 
simply  with  the  hands,  until  one  liter  is  obtained.  If  ^ 
smaller  amount  results,  sufficient  water  is  added  to  make  a 
liter.  This  meat-infusion  is  the  starting-point,  not  alone 
for  the  preparation  of  Loffler's  bouillon,  but  also  for  a 
whole  series  of  other  nutritive  media.  Instead  of  meat- 
infusion,  a  five  per  cent,  solution  of  Liebig's  meat-extract 
may  be  employed.  To  the  meat-infusion  are  added  ten 
grams  of  peptone  (one  per  cent.)  and  five  grams  of  sodium 
chlorid  (^  of  one  per  cent.),  and  the  whole  is  heated  in  an 
enameled  vessel  upon  an  open  fire,  until  the  peptone  and 
the  sodium  chlorid  have  been  dissolved.  At  the  same 
time  the  coagulable  albuminoid  substances  are  precipitated. 
The  coagula  floating  upon  the  surface  of  the  fluid  are  re- 
moved with  a  spoon,  and  the  fluid  itself  is  passed  through 
a  folded  filter  previously  moistened  with  distilled  water. 
The  fluid  obtained  should  be  perfectly  clear  and  of  acid 
reaction.  The  solution  is  now  rendered  slightly,  though 
distinctly,  alkaline  by  means  of  sodium  hydroxid,  and 
toward  the  close  preferably  with  sodium  carbonate.  In 
order  to  collect  the  precipitated  earthy  phosphates,  the  fluid 
is  further  heated  for  an  hour  in  the  autoclave  at  a  tempera- 
ture of  iio°  C.  (230°  F.),  or  in  the  steam-chest  for  two 
hours  at  a  temperature  of  100°  C.  (212°  F.).  Then  the 
still  warm  solution  is  filtered,  the  filtrate,  after  cooling,  again 
filtered,  and  made  up  to  a  liter  by  addition  of  distilled 
water.  The  finished  bouillon  should  be  amber-yellow  in 
color  and  transparent,  and  yield  a  feebly  alkaline  reaction. 
Should  the  reaction  be  not  alkaline,  it  must  absolutely  be 
made  so.  The  bouillon  is  now  introduced  into  the  test- 
tubes  closed  with  cotton  stoppers,  or  into  Erlenmeyer 
flasks,  which  are  best  sterilized  in  the  dry  chamber. 
Each  test-tube  will  contain  from  10  to  15  cu.  cm.,  each 
flask  from  50  to  100  cu.  cm.,  according  to  size.  Finally, 
the  tubes  or  flasks  containing  the  bouillon  are  sterilized  for 
an  hour  in  live  steam.  For  the  cultivation  of  special  bac- 
teria, additions  of  certain  substances  are  at  times  made  to 
the  bouillon — as,  for  instance,  grape-sugar,  in  proportion 
of  two  per  cent,  (grape-sugar  bouillon),  or  glycerin,  in  pro- 
portion of  from  four  to  six  per  cent,  (glycerin-bouillon). 
It  is  useful  to  have  constantly  in  readiness  a  sterile  ten  per 


80  CLINICAL  BACTERIOLOGY. 

cent,  solution  of  grape-sugar,  of  which  20  cu.  cm.  are 
added  to  each  Hter  just  before  introduction  into  the 
test-tubes,  in  order  to  Hmit  as  far  as  possible  the  caramel- 
ization  of  the  grape-sugar  that  occurs  on  protracted  heat- 
ing. Grape-sugar  bouillon  is  best  sterilized  by  exposure 
for  ten  minutes  in  the  steam-chest  on  three  successive  days. 
In  order  to  determine  whether  a  given  bacterium  generates 
acid,  a  few  drops  of  sterile  tincture  of  litmus  may  be  added 
to  the  bouillon. 

{b)  P reparation  of  Gelatin. — The  same  steps  are  fol- 
lowed as  in  the  preparation  of  bouillon,  except  that  to 
every  liter  of  meat-infusion  100  grams  (ten  per  cent.)  of 
gelatin  are  added  besides  ;  then  follow  solution  by  boiling 
in  the  steam -chamber,  alkalinization,  addition  of  the  white 
of  an  ^g^,  boiling  for  an  hour,  and  filtering.  In  the  case 
of  gelatin  also  the  reaction  must  be  tested  after  boiling. 
Gelatin  thus  prepared  remains  firm  up  to  temperatures  of 
about  24°  C.  (75.2°  F.).  If  gelatin  is  desired  that  remains 
firm  at  still  higher  temperatures  (27°  C. — 80.6°  F.,  28° 
C. — 82.4°  F.),  the  procedure  is  modified  by  reducing  the 
heat  applied  to  the  lowest  possible  minimum.  Forster  has 
determined  that  the  solidification-point  of  gelatin  is  reduced 
about  2°  for  every  hour's  heating.  The  most  practicable 
method  of  preparing  gelatin  is,  therefore,  as  follows  :  To  one 
liter  of  Loffler's  nutrient  bouillon  heated  in  a  vessel  over  a 
small  flame  to  about  60°  C.  (140°  F.)are  added  100  grams 
of  commercial  gelatin  cut  in  strips.  With  constant  stirring 
complete  solution  of  the  gelatin  is  effected  by  boiling  for 
seven  minutes.  Then  the  fluid,  rendered  acid  by  the  pres- 
ence of  the  gelatin,  is  carefully  alkalinized,  and,  after  addi- 
tion of  the  white  of  an  ^^^,  is  boiled  for  fifteen  minutes  in 
a  Papin  dish  ;  it  is  next  filtered  over  a  water-bath  at  a  tem- 
perature of  60°  C.  (140°  F.)  into  a  large  flask,  and  is  then 
distributed  among  test-tubes  (ten  cu.  cm.  to  each).  The 
test-tubes  are  next  sterilized  by  exposure  for  fifteen  minutes 
in  a  Papin  dish.  It  is  advisable  to  employ  a  perforated  tin 
shelf,  in  the  openings  in  which  the  tubes  may  be  placed  in- 
dividually, so  that  each  is  entirely  surrounded  by  water. 
To  expedite  the  process  of  heating,  this  shelf  is  at  first 
rotated  in  the  steam-cylinder.  The  nutrient  gelatin  thus 
obtained  is  completely  clear,  remains  firm  at  a  temperature 
of  27°  C.  (80.6°  F.)  or  28°  C.  (82.4°  F.),  and  is  always 
sterile.     It  may  be  permitted  to  harden  in  vertical  columns 


METHODS  OF   CULTURE  AND  OF  EXAMINATION.         81 

or  in  slanting  layers,  to  be  employed  subsequently  for  stab- 
cultures  or  for  streak-cultures.  The  liquefaction-point  of 
the  gelatin  may  be  increased  a  degree  or  two  if  it  be  per- 
mitted to  stand  for  twenty-four  hours  before  being  used 
(Forster). 

For  special  purposes  (cultivation  of  yeast-cells),  when 
gelatin  of  acid  reaction  is  required,  potato- gelatin  or  malt- 
gelatin  may  be  employed.  To  prepare  the  former,  500 
grams  of  cleansed,  peeled,  and  grated  potatoes  and  one 
liter  of  water  are  permitted  to  stand  together  for  three  or 
four  hours.  The  expressed  and  filtered  fluid,  the  potato- 
water,  is  sterilized  in  the  autoclave  for  an  hour  at  a  tem- 
perature of  110°  C.  (230°  F.),  or  for  fifteen  or  twenty 
minutes  at  a  temperature  of  120°  C.  (248°  F.),  and  then — 
instead  of  the  bouillon — is  used  for  the  preparation  of  the 
gelatin.  To  prepare  malt-gelatin,  the  beer-wort  or  infusion 
of  malt  that  can  be  obtained  in  any  brewery  is  sterilized, 
instead  of  potato-water,  and  is  then  further  treated  in  the 
same  way  as  the  bouillon.  The  only  difference,  as  com- 
pared with  the  preparation  of  simple  gelatin,  is  observed  in 
the  alkalinization,  only  so  much  normal  sodium  hydroxid 
being  added  as  will  restore  the  original  feebly  acid  reaction 
of  the  potato-water  or  of  the  malt-infusion  respectively. 

For  the  purpose  of  cultivating  typhoid-bacilli  directly 
from  the  feces,  Eisner  has  devised  a  modification  of  potato- 
gelatin  by  the  addition  oi  one  per  cent,  of  potassium  iodid. 
It  is  best  to  add,  by  means  ol  a  sterile  pipet,  ^  cu.  cm.  of 
a  germ-free  twenty  per  cent,  solution  of  potassium  iodid  to 
ten  cu.  cm.  of  potato-gelatin  directly  before  use. 

(c)  Preparation  of  Agar-agar. — Instead  of  gelatin,  from 
1.2  to  1.5  per  cent,  of  agar-agar  is  added  to  the  peptone- 
sodium-chlorid  meat-infusion.  Agar-agar  is  a  vegetable 
gelatin  derived  from  Japanese  and  East  Indian  seaweed. 
It  is  best  to  employ  for  this  purpose  powdered  agar-agar 
instead  of  that  in  strips,  as  the  latter  require  a  longer  time 
for  their  solution.  After  the  addition  of  from  five  to  ten 
grams  of  gum  arabic,  to  cause  the  agar  to  adhere  to  the 
surface  of  the  glass,  and  after  solution  of  the  mixture  in  the 
steam-chamber  for  two  or  three  hours,  the  fluid  is  rendered 
alkaline  in  the  usual  manner,  the  white  of  an  ^^^  is  added, 
heat  is  again  applied  for  one  hour,  and  the  solution  is  fil- 
tered. As  the  agar  undergoes  coagulation  at  39°  C. 
(102.2°  F.),  the  filtering  naturally  can  not  be  undertaken 
6 


82  CLINICAL  BACTERIOLOGY. 

at  ordinary  room-temperature.  It  is,  therefore,  best  to 
place  both  flask  and  funnel  in  the  steam-chamber  in  com- 
plete activity.  Even  under  these  circumstances  the  process 
of  filtering  occupies  several  hours.  The  nutrient  agar  may, 
further,  be  filtered  through  a  warm-water  funnel,  consisting 
of  a  copper  vessel  closed  by  a  lid  and  containing  a  metallic 
funnel  into  which  a  glass  funnel  can  be  introduced.  The 
metallic  funnel  is  covered  by  a  brass  lid  in  order  to  pre- 
vent evaporation.  By  means  of  a  thermo-regulator,  the 
water  contained  in  the  outer  filter  is  kept  constantly  at  a 
temperature  of  between  60°  C.  (140°.  F.)  and  70°  C.  (158° 
F.).  The  prepared  agar  is  introduced  into  test-tubes  in 
exactly  the  same  way  as  bouillon  and  gelatin,  and  it  is 
sterilized  by  boiling  for  an  hour  in  the  steam-chamber. 
Then  it  is  placed  in  a  slanting  position  in  order  to  obtain 
the  largest  possible  surface  for  inoculation,  or  it  is  permitted 
to  coagulate  vertically  for  the  culture  of  anaerobic  bacteria. 
In  the  process  of  solidification  the  agar  expresses  a  certain 
amount  of  water,  the  so-called  water  of  condensation. 

Gelatin  and  agar-agar  may,  like  bouillon,  be  modified  by 
the  addition  of  all  possible  substances  ;  thus,  grape-sugar 
two  per  cent.,  glycerin  from  four  to  six  per  cent.,  etc.  Gly- 
cerin-agar  in  particular  plays  an  important  part  in  bacteri- 
ologic  technic.  It  is  an  admirable  culture-medium,  suitable 
for  many  pathogenic  bacteria. 

For  special  purposes,  as  for  the  cultivation  of  gonococci 
and  influenza-bacilli,  the  preparation  of  blood-agar  is  to  be 
recommended.  By  means  of  a  platinum  loop  several  drops 
of  sterile  human  or  pigeon's  blood  are  smeared  upon  the  sur- 
face of  the  agar,  the  tubes  being  then  placed  in  the  thermo- 
stat for  one  or  two  days  at  a  temperature  of  37°  C.  (98.6° 
F.),  and  the  contaminated  ones  being  rejected.  Hemo- 
globin-agar  may  be  prepared  in  the  same  manner  by  smear- 
ing a  solution  of  hemoglobin  upon  the  surface  of  the  agar. 
The  solution  of  hemoglobin  is  prepared  in  the  following 
manner  :  Blood  obtained  with  aseptic  precautions  is  mixed 
with  an  excess  of  physiologic  salt-solution,  and  permitted 
to  stand  in  the  cold  for  twenty-four  hours.  The  resulting 
precipitate  of  red  blood-corpuscles,  with  water  in  not  too 
large  amount,  is  introduced  into  a  separatory  funnel  and  a 
like  quantity  of  ether  is  added.  The  mixture  is  well,  though 
not  too  vigorously,  shaken,  and  the  dark-red,  watery  solu- 
tion obtained  is  quickly  filtered.     A  drop  of  this  is  smeared 


METHODS   OF  CULTURE  AND  OF  EXAMINATION.        83 

upon    the   surface   of  the   agar  by  means   of  a  platinum 
needle. 

(d)  The  preparation  of  liquid  blood-serum  for  purposes 
of  culture  requires  a  rather  complex  procedure.  The  blood 
from  a  suitable  animal,  obtained  after  division  of  the  carotid 
artery  (in  slaughtering),  is  received  into  tall,  sterilized  glass 
cylinders,  and  is  permitted  to  stand  upon  ice  for  two  days, 
in  order  that  the  serum  may  completely  separate  from  the 
clot.  The  serum  is  then  distributed  among  disinfected  test- 
tubes  by  means  of,  sterilized  pipets,  and  placed  for  sev- 
eral days  in  the  thermostat  at  a  temperature  of  37°  C. 
(98.6°  F.).     By  this  means  it  is  rendered  certain  whether 


Fig.  13, — Flask    to  re-  Fig.  14. — Koch's  apparatus  for  coagulating  and 

ceive  blood-serum.  sterilizing  blood-serum. 

all  of  the  manipulations  have  been  made  without  contami- 
nation— that  is,  whether  the  serum  is  still  germ-free.  In 
this  event  the  serum  remains  perfectly  clear  in  the  thermo- 
stat. The  tubes  that  become  turbid  are  rejected.  In  order 
to  convert  the  liquid  blood-serum  into  a  solid  culture- 
medium,  the  tubes  are  placed  in  an  oblique  position  in  the 
Papin  steam-dish,  and  the  serum  is  permitted  rapidly  to 
solidify.  After  this  the  tubes  are  sterilized  by  exposure  for 
ten  minutes  on  three  successive  days  to  a  temperature  of 
100°  C  (212°  R). 

For  the  preparation  of  serum-plates  the  liquid  serum  is 
poured  into  sterilized  Petri  dishes  (Fig.  23),  and  is  then 
solidified  and  sterilized  in  the  same  way  as  the  serum  in 


84  CLINICAL  BACTERIOLOGY. 

test-tubes.  The  disturbing  water  of  condensation  can  be 
removed  from  the  surface  of  the  plates  by  placing  these  in 
an  inverted  position  for  forty-eight  hours  in  the  thermostat 
at  a  temperature  of  37°  C.  (98.6°  F.). 

Human  blood-serum  is  obtained  either  by  blood-letting 
or  from  placentas.  After  the  umbilical  cord  has  been 
ligated  and  divided,  the  maternal  extremity  is  disinfected  by 
means  of  mercuric  chlorid,  which  is  rinsed  off  with  distilled 
water.  An  incision  is  made  into  the  cord  above  the  point 
of  ligation,  and  the  blood  is  permitted  to  escape  into  steril- 
ized flasks.  The  conversion  into  culture-media  is  effected 
in  the  manner  already  described.  A  mixture  of  three  parts 
of  sheep's  blood-serum  with  one  part  of  a  one  per  cent, 
grape-sugar  bouillon  (Loffler's  blood-serurn)  may  be  em- 
ployed with  especial  advantage  for  several  purposes  (diag- 
nosis of  diphtheria),  being  solidified  and  sterilized  in  tubes 
or  plates  in  the  same  way  as  ordinary  blood-serum. 

Blood-serum  agar  (blood-serum  glycerin- agar),  which  also 
is  employed  with  advantage  for  special  purposes,  is  pre- 
pared by  adding  to  liquefied  agar  or  glycerin-agar  cooled 
to  40°  C.  (104°  F.)  an  equal  amount  or  half  the  quantity 
of  sterile  liquid  blood-serum  heated  to  40°  C.  (104°  F.). 
The  mixture  is  introduced  into  test-tubes  or  Petri  dishes, 
and  made  to  solidify  rapidly. 

[e)  The  Preparation  of  Potatoes. — Potatoes  constitute  an 
excellent  nutritive  medium  for  many  purposes.  They  may 
be  prepared  in  various  ways.  Large  potatoes  are  thor- 
oughly cleansed  with  a  brush  and  mercuric  chlorid,  are 
carefully  freed  of  their  peels  and  their  so-called  decayed 
spots  and  eyes,  and  are  cut  into  slices  about  one  cm.  thick, 
which  are  placed  in  glass  double  dishes.  The  peels, 
together  with  the  slices  of  potatoes,  are  exposed  for  an 
hour  to  steam  under  pressure  at  a  temperature  of  110°  C. 
(230°  F.),  or  for  fifteen  or  twenty  minutes  at  a  temperature 
of  120°  C.  (248°  F.) ;  or  suitable  oblique  pieces  are  cut 
out  of  the  potatoes  and  are  introduced  into  test-tubes  with 
some  cotton  at  the  bottom,  and  these  are  sterilized  in  the 
manner  already  described.  The  cotton  at  the  bottom  of 
the  tubes  is  for  the  purpose  of  absorbing  the  fluid  that 
appears  in  the  process  of  boiling,  and  with  its  aid  the 
potato  is  subsequently  kept  moist.  Roux  has  devised 
special  tubes  for  potato-cultures  which  are  narrowed  near 
the  bottom.      (Fig.  15.)     Upon  the  shoulder  or  projection 


METHODS   OF  CULTURE  AND  OF  EXAMINATION. 


85 


thus  formed  the  bits  of  potato  He.  The  lower  portion  of 
the  tube  is  filled  with  sterile  salt-solution  in  order  to  keep 
the  surface  of  the  potato  moist.  Instead 
of  salt-solution  other  fluids  are  used  for 
special  purposes.  Thus,  bits  of  potato 
that  just  dip  into  five  per  cent,  glycerin- 
solution  furnish  an  excellent  culture- 
medium  for  tubercle-bacilli. 

As  the  cut  surface  of  the  potato  is 
more  or  less  changed  by  protracted  ex- 
posure to  the  action  of  steam,  it  is  better 
to  boil  the  whole  potato,  and  then  to 
divide  it.  To  this  end  the  unpared 
potatoes  are  thoroughly  cleansed  by 
means  of  a  brush  and  a  solution  of 
mercuric  chlorid,  the  so-called  eyes  are 
cut  out,  and  sterilization  is  practised  for 
two  hours  in  the  steam-chamber  on  each 
of  two  successive  days,  or  more  simply 
and  better  for  an  hour  in  the  autoclave  at 
a  temperature  of  iio°  C.  (230°  F.),  or  for  twenty  minutes 
at  a  temperature  of  120°  C.  (248°  F.).  The  potatoes  must 
be  sterilized  thoroughly  because  they  are  often  the  seat  of 
special  bacilli  characterized  by  the  extraordinary  resistance 


Fig.  15.— Roux's  test- 
tube,  specially  designed 
for  potato-cultures. 


Fig.  16.— Method  of  slicing  potato  (after  Woodhead  and  Hare). 


of  their  spores  (red  potato-bacilli).  Then  the  hands  are 
thoroughly  disinfected  by  means  of  soap,  alcohol,  and 
mercuric  chlorid,  and  the  still  warm  potato  is  removed 
with  the   fingers   moist  with   mercuric   chlorid,  and   it   is 


86  CLINICAL  BACTERIOLOGY. 

divided  into  two  equal  parts  with  a  knife  sterilized  in  the 
flame.  The  halves,  resting  upon  sterile  rubber  rings,  are 
preserved  in  moist  culture-chambers,  whose  individual 
glass  parts  have  been   disinfected   with   mercuric   chlorid. 

(/)  Peptone-water. — For  the  diagnosis  of  cholera  a  solu- 
tion of  one  per  cent,  peptone  and  one  per  cent,  sodium 
chlorid  in  distilled  water  is  required.  This  is  rendered  alka- 
line, is  sterilized  for  an  hour  in  the  steam-chamber,  and  is 
kept  in  readiness  in  test-tubes,  or,  better,  in  so-called  Pas- 
teur flasks.  The  latter  are  closed  by  means  of  a  glass 
helmet  drawn  out  into  a  tube,  so  that  evaporation  of  the 
water  is  prevented,  and  they  provide  a  large  surface  of  fluid 
for  the  developing  bacteria. 

For  examinations  of  water,  and  also  for  the  cultivation 
of  cholera-vibrios  from  water,  a  twenty-five  per  cent,  solution 
of  peptone  and  of  sodium  chlorid  is  kept  in  readiness,  and 
is  preserved  in  test-tubes,  each  of  which  contains  four  cubic 
centimeters.  If  the  contents  of  such  a  tube  be  added  to 
lOO  cu.  cm.  of  the  water  to  be  examined,  a  one  per  cent, 
solution  of  peptone  and  of  sodium  chlorid  at  once  results, 
which,  after  being  rendered  alkaline,  is  known  as  a  fertiliz- 
ing solution  and  can  be  used  for  culture-purposes. 

i^g)  Milk  is  likewise  frequently  used  as  a  nutritive  medium. 
Fresh  milk  is  introduced  into  test-tubes  or  into  Erlenmeyer 
flasks  provided  with  cotton  stoppers  and  sterilized.  In 
order  to  exclude  bacteria  the  tubes  and  their  contents  are  ex- 
posed thrice  for  an  hour  each  time,  or  once  in  the  autoclave 
at  atemperature  of  iio°  C.  (230°  F.)  or  i2o°  C.  (248°  F.). 

(//)  To  determine  the  question  whether  a  given  variety 
of  bacteria  produces  acid  or  alkali  in  the  course  of  its  devel- 
opment, the  whey  of  Petruschky  may  be  employed  with 
advantage.  One  liter  of  milk  is  added  to  a  like  amount  of 
water,  and  coagulation  is  effected  by  adding  to  the  mixture 
the  smallest  possible  amount  of  acid  (acetic  acid).  The 
fluid  is  now  filtered,  and  the  filtrate  neutralized  with  sodium 
carbonate  and  heated  to  boiling.  By  this  means  the  acid 
reaction  is,  as  a  rule,  restored,  and  turbidity  results,  which 
is  removed  by  filtration.  Then  the  mixture  is  boiled  again, 
is  neutralized  with  sterile  sodium-carbonate  solution,  and 
sterile  tincture  of  litmus  is  added  until  a  faint  violet  color 
is  produced. 

(?)  Of  less  common  culture-media,  employed  only  for 
special  purposes,  may  be  mentioned  bread-pap  (dry  bread 


METHODS   OF  CULTURE  AND  OF  EXAMINATION.         87 

reduced  to  powder,  and  introduced  into  an  Erlenmeyer 
flask  with  enough  distilled  water  to  make  a  homogeneous 
soft  mass,  and  sterilized  by  exposure  thrice  for  an  hour  each 
time  in  the  steam-chamber),  which  is  used  especially  for  the 
cultivation  of  molds  ;  also  rice-pap  (Soyka)  (boiled  rice-milk 
sterilized  in  double  glass  dishes),  which  is  available  for  per- 
manent cultures;  infusion  of  hay,  decoction  of  prunes,  etc., 
which  may  be  employed  as  fluid  nutrient  media,  or  in  com- 
bination with  gelatin  or  agar. 

(y)  A  special  position  among  culture-media  is  occupied 
by  those  free  from  albuminoids.  The  constitution  of  that 
most  frequently  employed,  and  proposed  by  Uschinsky,  is,  as 
modified  by  Frankel,  as  follows  : 

Potassium  biphosphate, 2.0 

Sodium  chlorid, 5.0 

Ammonium  lactate,      6.0 

Asparagin, 4.0. 

The  mixture  is  dissolved  in  a  liter  of  water  and  is  ster- 
ilized. 

METHODS  OF  CULTURE. 

In  nature  and  in  the  products  of  disease  individual  varie- 
ties of  bacteria  are  but  rarely  found  isolated — that  is,  alone. 
Mostly,  several  species  are  found  together  in  the  material 
subjected  to  examination.  It  is  of  the  utmost  importance, 
in  the  process  of  investigation,  to  separate  from  one  another 
the  different  varieties  in  such  a  mixture  of  bacteria — that  is, 
to  isolate  them  in  pure  cidture.  This  has  been  rendered 
possible  by  the  bacteriologic  methods  introduced  by  Koch. 
The  principle  of  these  consists  in  inoculating  a  solid  culture- 
medium  that  has  been  liquefied  with  a  trace  of  the  bacterial 
mixture  to  be  examined,  distributing  this  as  well  and  as 
evenly  as  possible,  and  then  spreading  the  mixture  upon  a 
sterile  glass  plate.  The  liquid  nutrient  medium  becomes  solid 
again  at  room-temperature,  and  covers  the  plate  in  a  thin 
layer,  in  which  the  bacteria  now  develop,  but  not  indiscrim- 
inately among  and  next  to  one  another,  as  in  a  tube  with 
fluid  contents,  but  separated  considerably  from  one  another. 
At  every  point  where  a  bacterial  germ  becomes  fixed  in  the 
solidifying  mass  it  undergoes  isolated  development,  multi- 
plying and  forming  for  itself  a  special  colony.  By  this  means 
it  is  possible  to  observe  the  development  and  the  growth 
of  the  individual  germs  also  under  the  microscope,  and  to 


88  CLINICAL  BACTERIOLOGY. 

manipulate   further   the  colonies    of  a   single   species    of 
bacteria. 

The  particulars  of  Koch's  method  of  plate-making  are  as 
follows  :  With  the  aid  of  a  platinum  wire  melted  into  a 
glass  rod  (Fig.  17),  a  small  amount  of  the  material  to  be 
examined  is  introduced  into  a  gelatin-tube  whose  contents 


=<ito  — ~q^r 


Fig.  17. — Platinum  wire  swaged  into  brass  wire  and  reversible  for  transportation 
(as  devised  by  Dr.  J.  H.  McColloni) :  a,  Closed ;  b,  open;  c,  the  same  with  end  bent 
at  a  right  angle,  for  picking  up  colonies  in  test-tube ;  rf,  the  same  in  operation  (Ernst). 


have  previously  been  liquefied  by  immersion  in  water  at 
a  temperature  of  from  30°  C.  (%6^  F.)  to  40°  C.  (104°  F.). 
The  platinum  wire  must,  of  course,  have  been  heated  in  the 
flame  of  a  Bunsen  burner  or  of  a  spirit-lamp  before  being 
used,  in  order  to  be  freed  from  germs  that  may  have  been 
attached  to  it.  When  the  material  to  be  examined  contains 
many  bacteria,  it  is  sufficient  to  insert  the  extremity  of  the 
heated  wire,  but.  if  the  number  of  bacteria  is  small,  it  is 


Fig.  18. — Platinum  wire  twisted  into  a  loop. 

better  before  heating  the  wire  to  twist  it  into  a  loop  in  order 
that  it  may  take  up  more  of  the  material.  (Fig.  18.)  If  a  solid, 
compact  substance  is  to  be  examined  with  regard  to  the  pres- 
ence of  bacteria,  it  is,  by  means  of  a  glass  rod  sterilized  in 
the  flame  and  subsequently  cooled,  broken  up  in  a  watch- 
glass  similarly  treated ;  it  is  then  rubbed  up  with  sterile 


METHODS   OF  CULTURE  AND  OF  EXAMINATION. 


89 


bouillon  or  water,  and  a  small  amount  of  the  emulsion  is 
removed  with  the  heated  platinum  wire.  If  the  substances 
to  be  examined  are  particularly  tough,  it  is  well  to  render  a 
mortar  and  pestle  germ -free  by  dry  sterilization  or  by  ex- 
posure to  the  flame,  and  to  cover  the  mortar  with  sterile 
paper,  in  the  middle  of  which  is  an  opening  for  the  handle 
of  the  pestle.  The  material  is  now  rubbed  up  with  the 
pestle  until  a  thoroughly  homogeneous  emulsion  is  made, 
while,  at  the  same  time,  the  overlying  sheet  of  paper  pre- 
vents the  entrance  of  germs  from  the  air. 

The  tube  containing  the  liquefied  gelatin  is  taken  in  the  left 
hand  with  its  palm  directed  upward,  and  between  the  thumb 


Fig,  19.— Manner  of  holding  tubes  for  inoculation. 


and  the  index-finger ;  then,  with  a  slight  rotatory  move- 
ment, the  cotton  stopper  is  removed  with  the  right  hand,  and 
placed  with  its  free  extremity,  which  always  projects  from 
the  mouth  of  the  tube,  between  the  index-finger  and  the  mid- 
dle finger  of  the  left  hand.  (Fig.  19.)  The  platinum  needle, 
laden  with  the  well-divided  material  for  examination,  is 
introduced  into  the  nutrient  gelatin,  great  care  being  taken 
to  avoid  bringing  the  wire  into  contact  with  the  walls  of 
the  tube.  The  platinum  wire  is  withdrawn  and  immedi- 
ately sterilized  in  the  flame.  The  material  introduced  is 
admixed  with  the  liquefied  gelatin,  as  intimately  and  as 
evenly  as  possible,  by  means  of  repeated  shakings,  rotation, 
inclination,  and  sudden  straightening  of  the  tube,  now  again 


90  CLINICAL   BACTERIOLOGY. 

closed  with  a  cotton  stopper.  The  culture-medium  could 
now  be  poured  upon  a  plate  for  solidification,  but  this  is 
usually  not  done  at  this  time,  because,  as  a  rule,  the  first 
gelatin-tube,  or,  as  it  is  called,  the  original  tube,  contains 
too  large  a  number  of  bacteria,  whose  colonies  would  there- 
fore develop  too  closely  together  upon  the  plate.  For  this 
reason  a  first  and  a  second  dilution  are  yet  prepared.  To 
this  end  the  inoculated  original  tube  is  taken  between  the 
thumb  and  the  index-finger  of  the  left  hand,  another  gela- 
tin-tube, liquefied  at  40°  C.  (104°  F.),  is  held  between  the 
index-finger  and  the  middle  finger,  and  the  cotton  stoppers 


Fig.  20.— Manner  of  holding  tubes  during  inoculation :  a,  Original  tube ;  b,  tube  to 
be  inoculated  ;  c,  cotton  plugs. 

are  removed,  the  first  being  held  between  the  middle  and  the 
ring  finger,  and  the  second  between  the  ring  and  the  little 
finger.  Then,  with  the  platinum  needle  sterilized  by  heat, 
three  loopfuls  of  the  liquid  contents  of  the  original  tube 
are  successively  taken  from  the  original  tube  and  intro- 
duced into  the  second  gelatin-tube  (first  dilution).  (Fig.  20.) 
After  vigorous  shaking,  in  the  manner  described,  of  the 
tubes,  now  again  closed,  a  third  tube  of  hquefied  gelatin  is 
inoculated  in  an  identical  manner  with  three  loopfuls  of  the 
first  dilution  (second  dilution). 

Rectangular  plates  of  glass  will  meanwhile  have  been 
sterilized  in  sheet-iron  boxes  (plate-pockets)  in  the  hot-air 


METHODS  OF  CULTURE  AND  OF  EXAMINATION. 


91 


chamber,  and  one  of  these  plates,  grasped  carefully  at  its 
edges  with  the  fingers,  or,  better,  with  forceps  sterilized  in 
the  flame,  is  placed  upon  a  plate  covering  a  dish  filled  with 
ice.  Over  this  plate,  in  order  to  reduce  the  possibility  of 
aerial  infection  to  a  minimum,  is  placed  a  bell-glass  that 


Fig.  21. — Leveling  and  cooling  apparatus. 


has  been  immersed  in  mercuric-chlorid  solution.  The  entire 
apparatus  may  with  advantage  be  mounted  upon  a  leveling 
device.  (Figs.  21,  22.)  The  cotton  stopper  is  now  quickly 
removed  from  the  original  tube,  the  lips  of  which  are  passed 
through  the  flame  and  are  briefly  permitted  to  cool,  and 
after  removal  of  the  bell-glass  the  contents  of  the  tube  are 
poured  upon  the  center  of 
the  rectangular  plate,  and  are 
distributed  evenly  with  the 
lips  of  the  tube,  care  being 
taken  to  leave  a  free  space 
of  one  centimeter  at  the  bor- 
ders of  the  plate.  Some  of 
these  glass  plates  are  pro- 
vided, at  a  distance  of  one 
centimeter  from  their  bor- 
ders, with  a  wall  of  enamel 
about  one  millimeter  high, 
for  the  purpose  of  preventing 
overflow  of  the  gelatin.    The 

gelatin  solidifies  rapidly  upon  the  ice  base  after  the  bell- 
glass  is  replaced,  and  the  finished  plate  is  now  placed  upon 
a  glass  shelf  or  bench  in  a  crystallizing  dish,  which  is  con- 
verted into  a  moist  chamber  by  the  insertion  of  bibulous 
paper  moistened  with  a  solution  of  mercuric  chlorid.     The 


Fig.  22. — Complete  leveling  apparatus 
for  pouring  plate-cultures,  as  taught  by 
Koch. 


02 


CLINICAL   BACTERIOLOGY. 


first  and  the  second  dilution  are  poured  upon  plates  in  the 
same  way  ;  a  second  and  a  third  glass  shelf  placed  upon  the 
first  in  the  moist  chamber,  and  a  plate  respectively  placed 
upon  each  of  these. 

This  original  method  of  Koch  for  pouring  plates,  which 
is  still  used  in  certain  investigations,  has  been  replaced  by 
a  simpler  and  more  convenient  method  :  In  place  of  the 
plates  double  dishes  of  glass  are  used  (Petri  dishes,  Fig.  23). 
Sterilization,  liquefaction,  and  inoculation  of  the  tubes  are 
effected  exactly  in  the  same  way.  After  inoculation,  how- 
ever, the  liquefied  gelatin  of  the  three  tubes  is  simply 
poured  into  three  sterile  dishes,  which  are  then  covered  and 
left  to  themselves. 

In  accordance  with  the  bacteria  that  it  is  desired  to  cul- 
tivate, and  the  temperature  that  prevails  in  rooms,  the  fin- 
ished plates — that  is,  those  that  have  been  solidified — are 
placed  at  room-temperature  in  a  dark  place,  or   in    the 


Fig.  23. — Petri  dish  for  making  plate-cultures. 


thermostat  at  a  temperature  at  from  24°  C.  (75.2°  F.)  to 
26°  C.  (yS.S°  F.).  To  protect  the  plates  from  the  drying 
influence  of  the  air,  which  inhibits  the  growth  of  the 
bacteria,  it  is  useful  to  preserve  them  in  so-called  culture- 
boxes.  Each  of  these  consists  of  a  four-cornered  box  of 
sheet-iron  covered  with  a  glass  lid,  and  which  is  converted 
into  a  moist  chamber  by  the  introduction  of  a  dish  con- 
taining moistened  cotton  (Forster). 

The  method  described  suffices  when  all  that  is  desired  is 
the  determination  whether  bacteria  are  present  in  material 
submitted  for  examination,  as  well  as  their  nature.  If, 
however,  the  number  of  bacteria  present  is  to  be  accurately 
determined  also  quantitatively,  the  following  mode  of  pro- 
cedure is  adopted  :  Into  a  gelatin-tube  containing  exactly 
ten  cubic  centimeters,  after  liquefaction  a  given  amount  of 
the  material  to  be  examined  is  introduced  either  by  means 
of  a  sterile  pipet  (capillary  pipet)  or  with  a  platinum  loop 


METHODS   OF  CULTURE  AND  OF  EXAMINATION.         93 

(spiral  or  hook),  whose  capacity  has  been  determined  by- 
weighing.  Especial  importance  is  to  be  attached  in  this 
connection  to  an  equable  distribution  of  the  bacteria  by 
careful  admixture.  Of  the  mixture  exactly  the  same  care- 
fully measured  amount  is  introduced  into  ten  cubic  centi- 
meters of  gelatin,  and  then  of  this  the  same  amount  in  a 
third  tube  containing  ten  cubic  centimeters,  and  so  on. 
After  three,  four,  or  five  dilutions  have  been  made,  in 
accordance  with  the  approximate  number  of  bacteria 
originally  present  and  the  amount  of  inoculated  mate- 
rial, the  gelatin  is  poured  into  plates,  is  permitted  to 
solidify,  and  the  number  of  colonies  that  develop  upon  it 
in  the  course  of  the  next  eight  days  is  counted  daily. 
Generally  the  colonies  are  so  dense  upon  the  first  and 
perhaps  also  upon  the  second  plate,  that  their  enumeration, 


Fig.  24.— Wolff  hiigel's  apparatus  for  counting  colonies  of  bacteria  upon  plates. 

even  with  a  lens  or  with  a  low  power  of  the  microscope,  is 
not  possible.  If  the  dilutions  have  been  properly  made, 
there  will  always  be  one  plate  upon  which  the  colonies  can 
be  counted,  and  from  this  the  numerical  relations  of  the 
colonies  developed  upon  the  other  plates  can  readily  be 
estimated.  The  enumeration  is  greatly  facilitated  by  the 
use  of  a  dark  background  divided  into  square  centimeters, 
and  i  or  -i-  of  a  square  centimeter.  Such  an  instrument  is 
the  apparatus  of  Wolffhiigel  (Fig.  24),  which  consists 
essentially  of  a  glass  plate  divided  into  squares,  and  pro- 
vided with  a  dark,  dull  background. 

In  dealing  with  solid  substances,  i  gram,  ^^  gram,  etc., 
is  weighed,  rubbed  in  a  sterile  mortar  with  from  5  to  10 
cu.  cm.  of  sterile  bouillon  or  with  physiologic  salt-solution, 
and  the  remaining  steps  are  the  same  as  those  already 
described. 


94  CLINICAL  BACTERIOLOGY. 

If  it  is  desired  to  make  plates  of  agar-agar,  special  pre- 
cautions must  be  observed  in  consequence  of  the  ready 
coagulability  of  this  culture-medium  (even  at  39°  C. — 
102.2°  F.).  After  the  agar  has  been  liquefied,  best  in  boil- 
ing water  (it  melts  at  90°  C. — 194°  F.*),  the  tubes  are  placed 
in  a  water-bath  at  a  temperature  of  40°  C.  (104°  F.).  At 
this  temperature  the  agar  just  remains  liquid,  and  the  bac- 
teria can  be  inoculated  without  suffering  in  vital  activity. 
The  pouring  of  the  agar  in  the  double  dishes  is  unattended 
with  difficulty,  as  is  likewise  the  preparation  of  dilutions 
according  to  the  same  method  ;  only  it  is  necessary  to  be 
expeditious  in  the  execution  of  the  manipulations  in  order 
to  avoid  solidification  of  the  agar  in  the  tubes. 

The  use  of  the  gelatin-tube  itself  as  a  plate  is  rendered 
possible  by  Esmarch's  modification  of  the  Koch  procedure  : 
The  liquefied  tubes,  which  are  best  closed  with  a  plug  of 
nonabsorbent  cotton  not  freed  from  fat,  in  order  to  avoid 


Fig.  25.— Esmarch's   roll-culture :    a,  India-rubber  cap ;    b,  b,  b,   longitudinal   line 
drawn  on  the  tube  ;  c,  c,  c,  transverse  lines  for  counting  colonies  (Frankland). 


saturation  with  the  fluid,  are  inoculated,  closed  with  a  rub- 
ber cap,  held  in  ice-water,  and  rapidly  and  regularly  rotated 
about  its  axis.  The  gelatin  is,  by  this  means,  distributed 
in  a  thin,  even  layer  on  the  inner  surface  of  the  tube,  is 
solidified,  and  in  this  way  a  roll -plate  {¥\g.  25)  is  prepared, 
which  is  then  further  treated  in  the  same  way  as  ordinaiy 
plates. 

To  prepare  so-called  agar  streak-plates  for  rapid  diagnosis, 
the  liquefied  agar  is  poured  out  before  inoculation.  When 
it  has  solidified,  six  or  seven  strokes  are  made  side  by  side 
upon  an  agar-plate  with  a  platinum  needle  that  has  been 
dipped  in  the  fluid  to  be  examined,  or  with  the  material  to 
be  examined  itself  (membrane,  cotton  swab,  etc.).  Upon 
the  last  stroke  the  bacteria  will  be  so  sparsely  distributed 
that  individual  colonies  develop.  According  to  the  same 
principle  of  dilution,  the  surface  of  a  number  of  agar  or 
blood-serum    tubes    solidified    in    a    slanting   position    is 


METHODS   OF  CULTURE  AND  OF  EXAMINATION.         95 

rubbed  successively  with  the  inoculated  platinum  needle,  or 
similar  body  {^fractional  streak). 

The  finished  plates  are  kept  in  culture-boxes  for  from  two 
to  four  days  at  room-temperature,  or  in  the  thermostat 
at  a  temperature  of  24°  C  (75.2°  F.)  or  25°  C.  {tj""  F.) 
(gelatin-plates),  or  they  are  permitted  to  remain  in  the 
thermostat  for  from  twenty-four  to  forty-eight  hours  at  a 
temperature  of  37°  C.  (98.6°  F.)  {agar-plates).  After  the 
lapse  of  this  time  the  plates  are  examined  with  the  naked 
eye,  with  a  lens,  and  also  with-  a  low  power  of  the  micro- 
scope (from  50  to  100  magnifications).  It  is  next  deter- 
mined whether  one  or  several  varieties  of  colonies  have 
developed,  and  then  the  peculiarities  of  these  colonies — 
whether  they  liquefy  the  gelatin,  whether  they  possess  a 
sharp  or  an  irregular  border,  whether  they  are  granular, 
whether  they  present  a  definite  arrangement,,  special  tints 
of  color,  etc.  Variations  in  the  appearance  of  similar  col- 
onies are  dependent  upon  their  position  in  the  gelatin. 
Deeply  lying  colonies  almost  always  assume  a  spheric 
shape,  and  appear,  as  a  rule,  round  and  dark,  whereas  the 
superficial  colonies  sometimes  spread  like  a  membrane  upon 
the  surface  of  the  gelatin,  and  appear  bright  and  transparent. 
The  most  important  object  to  be  accomplished,  however,  is 
to  obtain  pure  cultures  from  these  plates.  If  the  particular 
colony  from  which  it  is  intended  to  make  inoculations  is 
not  entirely  too  small,  and  if  it  be  isolated,  the  procedure  is 
quite  simple  :  The  plate  or  the  dish  is  placed  upon  a  dark 
background,  the  platinum  wire  is  heated  in  the  flame,  and 
with  the  aid  of  the  naked  eye  or  of  a  lens  the  extremity 
of  the  wire  is  introduced  into  the  colony.  If,  however,  the 
colony  is  particularly  small,  there  is  no  alternative  but  to 
make  the  inoculation  with  the  platinum  wire  with  the  aid  of  a 
low  power  of  the  microscope.  For  this  purpose  a  steady  hand 
and  much  practice  are  required.  Apparatus  have  also  been 
devised  for  this  purpose,  but  they  are  rather  complicated,  and 
scarcely  more  trustworthy  for  the  experienced  manipulator. 
After  the  material  from  the  colony  has  been  taken  up  with 
the  needle,  it  is  smeared  upon  one  of  the  various  culture- 
media  after  having  convinced  oneself,  with  the  aid  of  the 
microscope,  if  necessary,  that  only  one  colony  has  been  re- 
moved, and  in  this  way  a  pure  culture  is  made.  In  inoc- 
ulating a  gelatin-tube  the  needle  is  generally  introduced 
from  above  downward  through  the  middle  of  the  column  of 


96 


CLINICAL  BACTERIOLOGY. 


gelatin.      In  this  way  a  gelatin  stab-culture  (Fig.  26,  a)  is 
obtained. 

Agar,  and  often  also  gelatin,  is  generally  solidified  so  as 
to  yield  a  slanting  surface  for  inoculation,  the  point  of  the 
needle  being  passed  from  below  upward  upon  the  surface 
o{^h^(z\x\\^lx^-\xv^6^^\m.  {streak-culture).  (Fig.  26,  <^.)  Inoc- 
ulations upon  potatoes  are  made  in  exactly  the  same  way. 

In  the  preparation  of  a  bouillon- 
culture  or  a  milk-culture  the 
mass  of  bacteria  is  rubbed  simply 
on  the  inner  surface  of  the  tube 
below  the  level  of  the  bouillon 
or  the  milk,  the  loop  used  for 
the  inoculation  being  then  thor- 
oughly shaken. 

In  order  to  inoculate  one  test- 
tube  from  another,  to  continue 
the  pure  culture  further,  the  two 
tubes  are  grasped  between  the 
fingers  (Fig.  20)  (the  first  tube 
between  the  thumb  and  the 
index-finger,  and  the  second 
tube  between  the  index-finger 
and  the  middle  finger),  their 
stoppers  are  removed  (the  one 
being  held  between  the  third  and 
fourth,  and  the  other  between 
the  fourth  and  fifth  fingers),  and 
from  the  first  tube  there  is 
taken,  with  the  platinum  needle 
previously  sterilized  in  the  flame, 
or,  in  the  case  of  fluid  culture- 
media,  with  the  platinum  loop,  a 
small  amount  of  the  culture, 
which  is  then  conveyed  upon  or 
into  the  new  culture-medium. 
The  reinoculation  of  pure  cultures  that  it  is  desired  to  main- 
tain must  be  repeated  at  intervals  of  four  weeks. 

The  development  of  gelatin-cultures  is  permitted  to  take 
place  at  room-temperature,  or  in  the  thermostat  at  a  tem- 
perature of  24°  C  (75.2°  F.)  or  26°  C.  (78.8°  F.).  The 
remaining  cultures  are,  however,  usually  kept  in  the  ther- 
mostat at  a  temperature  of  37°  C.  (98.6°  F.).     A  thermo- 


Fig.  26.— Stab-culture  (a) ;  streak- 
culture  {b). 


METHODS  OF  CULTURE  AND  OF  EXAMINATION. 


97 


Stat  or  incubator  consists  of  a  double-walled  copper  or  sheet- 
iron  chamber  surrounded  with  felt  or  asbestos.  The  space 
between  the  two  walls  is  filled  with  water,  which  is  kept  con- 
stantly at  an  equable  temperature  by  means  of  a  thermo-regu- 
lator.  The  layer  of  water  transmits  its  heat  to  the  interior  of 
the  thermostat,  the  actual  culture-chamber,  which  constantly 
should  have  a  temperature  of  37°  C. 
(98.6°  F.)  for  agar,  or  from  24°  C. 
(75.2°  F.)  to  26°  C.  (78.8°  F.)  for 
gelatin.  The  culture-tubes,  which 
are  to  be  kept  for  a  considerable 
length  of  time  in  the  thermostat, 
must  be  provided  with  rubber  caps 
sterilized  with  mercuric  chlorid,  in 
order  to  protect  them  from  evapora- 
tion. 

The  growth  in  a  number  of  these 
cultures  constitutes  for  many  bacteria 
a  definite  characteristic,  which  is  of 
the  highest  significance  in  their  iden- 
tification. Least  typical,  as  a  rule,  is 
the  growth  upon  agar.  Attention 
must  be  directed  to  the  thickness, 
the  transparency,  and  the  color  of 
the  deposit,  etc.    Jn  bouillon-cultures 

there  may  be  distinguished  bacteria  that  develop  only 
upon  the  surface  in  the  form  of  a  thick  or  a  thin  membrane, 
others  that  render  the  bouillon  more  or  less  homogene- 
ously turbid,  and  still  others  that  grow  only  at  the  bottom 
as  a  crumbling  or  a  viscid  sediment.  Milk  is  unaltered 
by  many  bacteria,  while  others  cause  coagulation  through 
the  formation  of  acid.  The  most  important  peculiarities 
are  furnished  by  gelatin-cultures.  In  the  first  place  the 
liquefaction  that  results  through  the  activity  of  a  peptoniz- 
ing ferment  is  to  be  looked  for.  This  occurs  in  part  only 
superficially,  and  is  in  part  funnel-shaped  or  stocking-shaped 
at  a  depth.  If  liquefaction  does  not  take  place,  varied 
and  often  characteristic  peculiarities  of  growth  occur  (nail- 
culture,  tree-like  division,  etc.).    " 


Fig.  27. — Method  of  inocu- 
lating a  test-tube  containing 
sterile  nutrient  jelly. 


CLINICAL  BACTERIOLOGY. 


CULTIVATION  OF  ANAEROBIC  BACTERIA. 

Anaerobic  bacteria  may  be  facultative  or  obligate.  The 
former  may  develop  also  in  the  presence  of  oxygen, 
although  but  sparingly.  The  latter  require  special  cultural 
methods.  The  cultivation  of  strictly  anaerobic  bacteria  is 
undertaken  either  in  a  room  free  from  air,  or  in  an  atmos- 
phere of  indifferent  gas — as,  for  instance,  hydrogen — or 
with  the  employment  of  substances  that  absorb  oxygen,  or, 
finally,  by  means  of  stab-culture  in  a  high  layer.  The 
usual  culture-media  are  employed,  but  with  the  addition  of 


Fig.  28. — Frankel's  method  of  making 
anaerobic  cultures. 


Fig.  29. — Hesse's  method  of  making 
anaerobic  cultures. 


two  per  cent,  of  grape-sugar,  as  all  of  the  anaerobic  bacteria 
thus  far  known  form  from  this  substance  gas  in  abundance 
(carbon  dioxid,  hydrogen  sulphid,  methane,  mercaptan, 
etc.),  in  this  way  displacing  the  oxygen  of  the  air. 

Plate-cultures. — According  to  a  method  devised  by  R. 
Koch,  a  sheet  of  mica,  sterilized  by  heat,  is  placed  upon  the 
liquid  gelatin  spread  upon  the  plate.  After  solidification  has 
taken  place  the  gelatin  is  thus  rendered  air-tight,  and  be- 
neath the  mica  the  anaerobic  colonies  undergo  development. 
It  is  more  serviceable  in  the  preparation  of  anaerobic  plates  to 


METHODS  OF  CULTURE  AND  OF  EXAMINATION. 


99 


employ  special  culture-dishes,  which  permit  the  entrance  of 
hydrogen  through  two  openings  in  the  lid  that  communicate 
with  a  gutter-like  excavation  of  the  dish.  The  lid  is  fas- 
tened to  the  periphery  of  the  dish  by  means  of  vaselin,  and 
it  is  revolved  as  soon  as  the  vessel  is  filled  with  hydrogen, 
in  order  that  the  openings  in  the  lid  and  the  gutter  are  no 
longer  opposed  to  one  another,  and  communication  with 
the  outside  is  cut  off  (Kamen  dish).  The  hydrogen  is  gen- 
erated in  a  Kipp's  apparatus  (Fig.  30)  that  is  filled  with 
pure  zinc  and  sulphuric  acid,  and  is  freed  of  hydrogen 
sulphid  and  of  oxygen,  by  means  of  two  wash-bottles  con- 


Fig.  30. — Kipp's  apparatus    for   producing   hydrogen,  with  wash-bottles   attached 

(Ernst). 


taining  an  alkaline  lead-solution  and  an  alkaline  pyrogallic- 
acid  solution.  The  employment  of  Kamen's  plates  is 
attended  with  certain  difficulties ;  especially  is  it  difficult  to 
drive  out  all  of  the  oxygen. 

In  the  preparation  of  anaerobic  plates  in  an  atmosphere 
of  hydrogen  Botkin's  apparatus  (Fig.  3 1)  is  employed.  This 
consists  of  a  large  bell-jar,  within  which,  upon  a  glass  stand, 
plates  are  exposed  free,  without  a  cover.  The  bell  stands 
upon  a  lead  cross  in  a  large  glass  dish.  Between  the  mar- 
gin of  the  bell  and  the  stand  is  a  space  through  which 
passes  a  U-shaped  rubbeir  ti^be  for  the  conduction  of  the 


100 


CLINICAL  BACTERIOLOGY. 


hydrogen  gas  into  the  upper  portion  of  the  bell.  Perfect 
closure  is  effected  with  the  aid  of  liquid  paraffin.  The  dis- 
placed air  escapes  at  the 
bottom  through  a  second 
rubber  tube,  which  is  re- 
moved after  the  apparatus 
has  been  completely  filled. 
Beneath  the  glass  bell  a 
vessel  containing  an  alka- 
line solution  of  pyrogallic 
acid  is  placed  for  further 
security.  A  disadvantage 
of  Botkin's  apparatus  con- 
sists in  the  fact  that  the 
plates  are  but  incompletely 
protected  against  contami- 
nation by  the  air. 

Novy's   apparatus    is   to 
be    warmly   recommended, 
both    for  plate-cultures   as 
well    as   for   test-tube  cul- 
tures.    This    consists  of  a 
high    glass    jar    upon    the 
edges  of  which  an   air-tight  helmet-like  cover  is  placed. 
The    latter   is    provided  above   with   a  revolving,    doubly 
perforated  glass  stopper,  through  which  the  hydrogen  gas 


Fig.  31. — Botkin's  apparatus  for  making 
anaerobic  plate-cultures. 


^ 


Fig.  32.— Novy's  jars  for  anaerobic  cultures. 


is  introduced  and  the  air  is  at  the  same  time  expelled.     At 
the  close  of  the  Qpe^atiqn  the  ;stopper  Js^  ^si^lp>y  rotated,  and 


METHODS  OF  CULTURE  AND  OF  EXAMINATION.       101 


the  inner  space  is  cut  off  completely  from  the  outer  air. 
(Fig.  32.)  Novy's  apparatus  may  also  be  connected  with 
an  air-pump,  and  be  exhausted  of  air  ;  or,  finally,  it  may  be 
freed  of  oxygen  with  the  aid  of  an  alkaline  solution  of 
pyrogallic  acid.     (See  method  of  Buchner,  below.) 

Aiiaerobic  pure  cultures  in  test-tubes  may  be  prepared  in 
the  following  manner  : 

1.  In  a  High  Layer. — Stab-cultures  are  made  in  tubes 
that  are  filled  with  nutrient  material  to  a  higher  level  than 
usual,  and  that  are  boiled  again  shortly  before  being  used. 
In  the  lower  portions  of  the  culture,  free  from  oxygen,  to 
which  the  needle  has  penetrated,  development  takes  place, 
whereas  the  upper  portions  of  the  medium  containing 
oxygen  remain  sterile.  The  anaerobic  bacteria  thrive  more 
vigorously  when  reducing  substances  are  added  to  the 
nutrient  media — e.  g.,  two  per  cent,  of  grape-sugar  or  from 
0. 3  to  o.  5  per  cent,  of  formic  acid. 

2.  In  an  Atmosphere  of  Hydrogen. — The  test-tubes  are 
closed  with  sterilized  rubber  stoppers  doubly  perforated, 
through  which  two  glass  tubes  bent  at  a  right  angle  lead  into 
the  interior.  (Fig.  28.)  Through  the  longer 

tube,  which  reaches  almost  to  the  nutrient 
medium  and  is  closed  with  a  cotton  stop- 
per, hydrogen  is  passed,  while  through  the 
shorter  tube  atmospheric  air  is  expelled. 
As  soon  as  gas  in  pure  state  escapes 
through  the  shorter  arm,  both  tubes  are 
sealed  by  heat,  or  closed  by  means  of  rub- 
ber tubes.  Inoculation  must,  of  course, 
have  been  effected  before  the  introduction 
of  the  hydrogen. 

J.  With  Complete  Exclusion  of  Air. — A 
test-tube  with  a  long-drawn-out  tenuous 
neck  is  filled  with  nutritive  material  and 
inoculated  in  the  usual  manner.  The  neck 
is  then  connected  with  an  air-pump,  and 
when  the  air  has  been  completely  ex- 
hausted, the  tube  is  sealed  by  heat. 

4.  According  to  the  Method  of  Buchner. — 
The  culture-tubes,  closed  by  means  of  a 
loosely  introduced  cotton  stopper,  are  in- 
troduced into  a  large,  hermetically  sealed  tube,  whose  floor 
is  covered  with  alkaline  solution  of  pyrogallic  acid  ( i  gram 


Fig-  33' — Buchner's 
method  of  making 
anaerobic  cultures. 


102 


CLINICAL  BACTERIOLOGY. 


of  pyrogallic  acid,  lo  cu.  cm.  of  i  per  cent,  potassium 
hydroxid).  (Fig.  33.)  Pyrogallic  acid  has  the  peculiarity 
of  taking  up  oxygen,  and  in  this  way  the  space  in  which 
the  cultures  are  exposed  is  quickly  freed  of  oxygen. 

Raw  eggs  also  may  be  employed  for  anaerobic  culture. 
One  extremity  of  the  ^^^  is  thoroughly  cleansed  with 
mercuric  chlorid  and  sterile  water,  a  puncture  is  made  with 
a  needle  sterilized  in  the  flame  and  still  hot,  and  with  the 
platinum  needle  a  portion  of  the  pure  culture  is  introduced 
into  the  interior  of  the  ^^'g.  The  small  opening  made  is 
then  closed  with  hot  sealing-wax. 

MICROSCOPIC   EXAMINATION  AND  STAINING  OF   BACTERIA. 

To  examine  bacteria  in  the  living  state  in  the  hanging 
drop,  a  small  drop  is  removed  from  a  fluid  culture  by 
means  of  the  platinum  loop  sterilized  in  the  flame,  and  it  is 
then  placed  in  the  center  of  a  cover-slip  carefully  cleansed 
with  alcohol.    (.Fig.  34.)    The  cavity  of  a  slide  that  has  been 


0 

Fig.  34. — The  "  hanging  drop"  seen  from  above  and  in  profile  (Mallory  and  Wright). 


excavated  at  its  center  is  surrounded  with  vaselin,  and 
the  slide  is  pressed  face  down  upon  the  cover-slip,  so  that 
the  drop  is  suspended  exactly  in  the  center  of  the  cavity. 
If  it  be  desired  to  prepare  a  hanging  drop  from  a  solid 
culture,  a  drop  of  sterile  water  or  of  bouillon  is  first  placed 
upon  the  cover-slip,  and  to  this  is  added  a  minimal  amount 
of  the  bacteria  to  be  examined.  It  is  useful  in  some  cases 
to  add  to  the  hanging  drop  a  small  amount  of  a  dilute 
staining  solution  :  e.  g.,  2l  carbol-fuchsin  solution  (p.  104) 
diluted  four  or  five  times.  The  small  amount  of  coloring- 
matter  added  does  not  influence  the  vital  activity  of  the 
bacteria.     These  are  stained  faintly,  but  the  motile  varieties 


METHODS  OF  CULTURE  AND  OF  EXAMINATION.       103 

continue  in  active  movement,  and  in  many  of  them,  as  a 
result  of  the  action  of  the  stain,  even  the  flagella  become 
visible.  In  making  examinations  in  hanging  drop  it  is 
advisable  first  to  find  the  border  of  the  drop  with  a  low 
power  of  the  microscope,  and  then  to  scrutinize  it  with 
higher  powers.  After  having  observed  sufficiently  the  size 
and  the  shape  of  the  bacteria  in  a  thin  layer,  and  while 
comparatively  quiet,  the  central  portions  of  the  drop  are 
brought  into  the  field. 

Instead  of  a  simple  hanging  drop,  a  culture  may  be  made 
in  hanging  drop,  and  the  growth  of  the  individual  bac- 
terial cell,  the  formation  of  spores  and  their  germination, 
etc.,  can  be  observed  directly  under  the  microscope.  As 
a  matter  of  course  the  cover-slip  must  previously  have 
been  sterilized  by  being  passed  through  the  flame.  After 
the  slip  has  cooled,  a  drop  of  sterile  bouillon  or  gelatin  is 
placed  at  its  center  and  is  inoculated.  The  observation 
may  then  be  continued,  perhaps  with  the  aid  of  a  warm 
stage,  or  of  a  special  small  incubator  in  which  the  entire 
microscope  is  introduced. 

In  order  to  decide  with  certainty  whether  the  movement 
observed  in  certain  bacteria  is  dependent  upon  their  own 
motility,  upon  molecular  movement,  or  upon  that  due  to 
currents,  it  is  at  times  necessary  to  employ  liquid  gelatin  as. 
a  diluting  fluid  instead  of  sterile  water.  The  degree  of 
temperature  necessary  for  liquefaction  is  readily  obtained 
by  using  the  warm  stage.  All  such  observations  are,  of 
course,  made  through  a  narrowed  diaphragm. 

Examination  of  Stained  Preparations. — For  staining 
purposes  basic  aniline  dyes  are  employed  that  possess  the 
property  of  staining  nuclei  and  bacteria.  The  most  impor- 
tant of  those  in  use  are  gentian -violet,  methyl -violet,  fuch- 
sin,  methylene-blue,  vesuvin,  and  malachite-green.  These 
stains,  with  the  exception  of  the  last  two,  are  kept  in  readi- 
ness in  concentrated  alcoholic  solution,  and  the  two  ex- 
cepted, vesuvin  and  malachite-green,  in  about  one  per  cent, 
watery  solution.  From  the  alcoholic  stock-solutions  the 
usual  watery  staining  solutions  are  prepared  by  dilution 
with  from  ten  to  twenty  times  the  amount  of  distilled  water. 
A  still  simpler  means  consists  in  permitting  a  few  drops  of 
the  stock -solution  to  pass  through  a  filter  into  a  watch-glass 
containing  distilled  water.  By  the  addition  of  certain  sub- 
stances that,  to  a  certain  extent,  play  the  part  of  mordants, 


104  CLINICAL  BACTERIOLOGY. 

the  staining  power  of  these  substances  may  be  increased 
considerably.  Among  substances  to  be  mentioned  in  this 
connection  are : 

1.  Potassium  hydroxid  (employed  by  Loffler  in  combina- 
tion with  methylene^blue)  : 

30  cu.  cm.  of  concentrated  alcoholic  solution  of  methylene-blue. 
ICX)  cu.  cm.  of  o.oi  per  cent,  solution  of  potassium  hydroxid  (i  :  10,000). 

This  so-called  Loffler's  solution  stains  well,  and  may  be 
preserved  for  an  almost  indefinite  time. 

2.  Aniline  water : 

Four  or  5  cu.  cm.  of  aniline  oil,  with  100  cu.  cm.  of  dis- 
tilled water  (i  part  of  aniline  oil  to  about  20  parts  of 
water),  are  vigorously  shaken  and  passed  through  a  moist- 
ened filter.  To  the  clear  filtered  aniline  water  (100  cu.  cm.) 
are  added  1 1  cu.  cm.  of  a  concentrated  alcoholic  solution 
of  fuchsin  or  of  gentian-viplet  or  methyl-violet.  It  is  more 
convenient  to  filter  the  aniline  water  in  a  watch-glass  and  to 
add  the  solution  of  fuchsin  or  gentian-violet  or  methyl- 
violet  until  a  metallic,  opalescent  pellicle  appears  upon  the 
surface.  These  solutions  of  Ehrlich  stain  well,  but  they 
possess  the  disadvantage  that  they  decompose  rapidly,  and 
they  must,  therefore,  be  freshly  prepared  each  time  that 
they  are  used. 

J.    Carbolic  acid: 

One  gram  of  fuchsin  is  dissolved  in  10  cu.  cm.  of  96  per 
cent,  alcohol,  and  then  90  cu.  cm.  of  5  per  cent,  carbolic 
acid  are  added. 

This  solution  of  Ziehl  does  not  possess  quite  so  intense  a 
staining  power  as  the  aniline-water  solutions,  but  it  has  over 
these  the  not  inconsiderable  advantage  of  greater  durability. 
In  an  analogous  manner  carbolic-acid  gentian-violet,  and 
carbolic-acid  methylene-blue  solutions  are  prepared,  both 
of  which  possess  excellent  staining  qualities,  and  can  be 
preserved  for  a  long  time. 

Preparation  and  Staining  of  Cover-glass  Specimens. — 
In  making  stained  preparations  the  suspected  material  is 
spread  in  as  thin  a  layer  as  possible  upon  a  cover-glass 
thoroughly  cleansed  with  alcohol  and  dried,  a  small  drop 
being  taken,  by  means  of  the  platinum  loop,  directly  from 
liquid  cultures  not  more  than  one  or  two  days  old,  or  a 
small  amount  of  the  bacterial  mass  from  solid  cultures  being 
rubbed  up  in  a  drop  of  sterilized  water  (or  tap-water,  which 


METHODS  OF  CULTURE  AND  OF  EXAMINATION.       105 

may  be  considered  sufficiently  sterile  for  this  purpose)  upon 
the  cover-glass.  The  preparation  is  permitted  to  dry  in  the 
air,  and  then,  in  order  to  fix  it,  it  is  passed  three  times, 
with  moderate  rapidity,  through  the  flame  of  a  spirit-lamp 
or  of  a  Bunsen  burner.  In  order  to  obtain  satisfactory 
preparations  from  cultures  it  is  useful,  before  staining,  to 
immerse  the  cover-glasses  for  from  one-half  to  one  minute 
in  from  a  one  to  a  four  per  cent,  solution  of  acetic  acid.  By 
this  means  the  preparation  is  cleared,  while  the  bacteria  in 
no  wise  suffer.  The  acetic  acid  is  blown  off  by  means  of  a 
glass  tube,  or  the  cover-slip  is  simply  dried  carefully  be- 
tween filter-paper.  By  means  of  a  pipet  or  of  a  small  filter 
(it  is  usually  well  to  filter  the  staining  solutions  before  they 
are  used)  several  drops  of  the  staining  solution  are  placed 
upon  the  smeared  surface  of  the  cover-slip  (conveniently 
grasped  with  Cornet's  forceps)  ;  the  stain  is  permitted  to 
exert  its  influence  for  a  short  time,  and  is  then  washed  off 


Fig-  35- — Stewart's  cover-glass  forceps. 

in  water.  If  the  staining  solution  has  been  placed  in  a 
watch-glass,  the  cover-slip  is  carefully  placed  upon  the  sur- 
face of  the  fluid  so  that  it  floats  thereon  with  its  smeared 
surface  down.  As  to  the  length  of  time  that  the  cover- 
glasses  should  be  exposed  to  the  influence  of  the  staining 
solution,  no  definite  statement  can  be  made.  This  will  de- 
pend upon  the  thickness  of  the  layer,  the  concentration  of 
the  stain,  and  the  tingibility  of  the  bacteria.  In  general, 
from  half  a  minute  to  a  minute  is  required  for  staining.  .  The 
time  of  exposure  to  the  action  of  the  staining  fluids  may  be 
materially  shortened  by  application  of  heat.  For  this  pur- 
pose the  cover-glass  is  held  with  the  forceps  directly  over 
the  flame  until  the  vapor  of  steam  is  given  off  from  the 
staining  solution.  The  stained  specimen  is  thoroughly 
washed  in  water,  then  carefully  dried  between  filter-paper, 
and  mounted  in  xylol-Canada  balsam.  If  blood-preparations 
are  to  be  examined  for  bacteria,  it  is  likewise  well  to  treat 
the   smeared    cover-glasses  according    to  the  acetic-acid 


106  CLINICAL  BACTERIOLOGY. 

method,  as  by  this  means  the  blood  coloring-matter  and  a 
portion  of  the  plasma  are  removed. 

The  specimens  prepared  in  the  manner  described  show 
the  size  and  the  shape  of  the  bacteria,  but  fail  to  disclose 
their  mutual  positions  in  the  colonies.  It  this  information 
be  desired,  so-called  contact-preparations  or  impress-prepa- 
rations must  be  made.  A  carefully  cleaned  cover-slip  is 
lightly  applied  or  impressed  upon  an  isolated  bacterial 
colony  of  a  gelatin-plate  or  an  agar-plate,  and  immediately 
removed.  After  it  has  been  dried,  this  preparation  is  further 
treated  exactly  in  the  manner  described. 

Staining  of  Sections  of  Tissue. — Bits  of  tissue  hard- 
ened according  to  the  usual  methods  and  embedded  in  cel- 
loidin  are  cut  with  the  aid  of  the  microtome.  The  sections 
are  next  placed  in  distilled  water,  and  from  this  are  trans- 
ferred to  one  of  the  staining  solutions  mentioned.  In  this 
they  are  permitted  to  remain  in  general  from  two  to  fifteen 
minutes.  The  excess  of  coloring-matter  is  removed  by 
means  of  dilute  acetic  acid  (i  :  looo),  and  the  sections  are 
rinsed  in  water  for  the  removal  of  the  acid,  dehydrated  with 
absolute  alcohol,  cleared  with  xylol,  and  mounted  upon  a 
slide  in  xylol-Canada  balsam.  It  is  advisable  first  to  spread 
the  sections  upon  slides,  and  to  undertake  the  staining, 
the  decolorization,  the  dehydration,  and  the  clearing  upon 
these.  On  the  whole,  this  mode  of  procedure  agrees  with 
that  originally  described  by  Weigert.  If  the  tissue  has 
been  embedded  in  paraffin,  this  must  be  extracted  with 
xylol  before  staining,  and  the  xylol  be  then  removed  with 
alcohol. 

The  methods  of  staining  thus  far  described  have  the 
property  of  staining  all  bacteria  in  the  same  way.  There 
is,  however,  a  method  that  stains  some  of  the  bacteria 
specifically — that  is,  Gram's  method. 

Gram's  Method. — The  prepared  cover-slips  from  a  fresh 
culture  from  twenty-four  to  forty-eight  hours'  old  are 
placed  in  aniline-water  gentian -violet  solution  (cover-glasses 
for  one  or  two  minutes,  sections  directly  out  of  alcohol  for 
ten  or  fifteen  minutes),  and  then  for  half  a  minute,  or  for  two 
and  a  half  minutes  or  three  minutes,  in  an  iodin  potassium- 
iodid  solution,  consisting  of  iodin  i.o,  potassium  iodid  2.0, 
water  300.0.  Decolorization  is  next  effected  in  alcohol,  and 
is  continued  as  long  as  any  color  is  given  off.  The  prepara- 
tion, which  finally  appears  colorless  (light  gray),  is  then 


METHODS  OF  CULTURE  »l«PjQF-SgCAMINATl€«S. Jj07 

X^/n;--    .-.,.. •'"^ 

rinsed  in  water,  dried,  and  niounterM4,^0att^"d^  j45ali 
Stained  in  this  way  certain  bacteria  assumed  Dltiislf^lack 
color,  while  others  yield  up  the  stain  completely  on  decol- 
orization.  Gram's  method,  which  is  of  especial  importance 
in  the  identification  of  bacteria,  is  most  difficult  of  applica- 
tion. The  slightest  variations  in  its  technic  may  render  the 
results  doubtful.  It  is  well,  therefore,  to  make  a  control- 
stain  in  every  instance,  examining  simultaneously,  in 
addition  to  the  bacteria  under  investigation,  another  variety 
of  bacteria  that  with  certainty  either  stains  (staphylococci) 
or  decolorizes  (bacterium  coli). 

Giinther's  Modification  of  Gram's  Method. — After 
exposure  to  the  action  of  the  iodin  potassium-iodid  solution, 
the  specimen  is  placed  for  half  a  minute  in  alcohol,  then  for 
a  short  time  (ten  seconds)  in  three  per  cent,  hydrochloric- 
acid  alcohol,  and,  finally,  for  complete  decolorization,  in 
pure  alcohol.  Before  mounting,  the  sections  are  cleared  in 
oil  of  cloves  or  in  xylol. 

Weigert's  Modification  of  Gram's  Method. — After 
treatment  with  aniline-water  gentian -violet  solution,  the 
specimen  is  rinsed  in  water,  and  the  section  is  spread  upon 
a  slide,  iodin  potassium-iodid  is  added,  and  then  removed 
with  bibulous  paper.  Next  follow  differentiation,  dehydra- 
tion, and  clearing  with  aniline  oil  and  xylol,  and  mounting 
in  Canada  balsam. 

Double  Staining. — To  make  a  contrast  between  the 
bacteria  and  the  surrounding  tissue,  it  is  advisable,  after 
staining  by  Gram's  method,  to  stain  the  sections  further 
with  a  dilute  watery  solution  of  vesuvin,  safranin,  or  car- 
mine, and  to  rinse  in  alcohol.  In  this  way,  however,  the 
bacteria  under  some  circumstances  lose  a  portion  of  their 
stain.  For  this  reason  it  is  preferable  to  apply  the  contrast- 
stain  before  using  Gram's  method  of  staining  the  bacteria. 
To  this  end  the  sections  are  washed  in  water,  stained  in 
picrocarmine  solution,  again  washed  in  water  and  placed  in 
alcohol,  after  which  the  staining,  according  to  the  method 
of  Gram,  can  be  undertaken  at  once  or  at  any  convenient 
time.  The  staining  of  the  tissues  is  in  no  way  interfered 
with  by  Gram's  method.  Not  all  bacteria  remain  stained 
after  application  of  Gram's  method :  a  very  consider- 
able number  can  not  be  demonstrated  by  this  means. 
Particulars  in  this  regard  will  be  found  in  the  special 
section. 


108  CLINICAL  BACTERIOLOGY. 

Staining  of  Capsules. — Some  bacteria  are  character- 
ized by  the  formation  of  capsules  in  the  blood  and  in 
other  animal  substances  (anthrax-bacilli,  pneumococci,  ba- 
cillus of  Friedlander,  etc.).  Occasionally,  the  capsules 
appear  also  in  cultures,  and  their  demonstration  can  be 
made  in  the  following  method  of  Johne  :  The  preparations 
are  stained  in  a  warmed  two  per  cent,  solution  of  gentian- 
violet,  are  rinsed  in  water,  decolorized  for  from  ten  to 
twenty  seconds  in  two  per  cent,  acetic  acid,  and  are  washed 
and  mounted  in  water  (not  Canada  balsam,  as  this  causes 
the  capsules  to  shrink). 

Staining  of  Spores. — Spores  are  stained  with  extraor- 
dinary difficulty,  because  of  the  dense  and  impenetrable 
membrane  by  which  they  are  surrounded.  The  usual 
methods  of  staining  are  not  sufficient  for  the  staining  of 
spores.  This  may  be  effected  by  exposure  of  cover-glass 
specimens  prepared  in  the  usual  way  with  spore-containing 
bacteria  for  an  hour  in  hot  carbol-fuchsin  solution  or  aniline- 
water  fuchsin-solution,  which  is  from  time  to  time  brought 
to  the  boiling-point.  By  addition  of  solution  undue  con- 
centration is  avoided.  The  specimen  is  taken  directly  from 
the  stain,  and  is  placed  in  ten  per  cent,  hydrochloric  acid 
or  in  three  per  cent,  hydrochloric-acid  alcohol,  in  which  it 
is  washed  for  about  a  minute.  By  this  means  everything 
is  decolorized  with  the  exception  of  the  spores,  which  re- 
tain their  stain.  It  is  here,  also,  useful  to  make  a  contrast- 
stain,  by  exposing  the  specimen  briefly  to  the  action  of  a 
watery  solution  of  methylene-blue  or  of  malachite -green. 
The  bacilli  then  appear  blue  or  green  respectively,  and  the 
spores,  on  the  contrary,  a  bright  red. 

If  it  be  desired  to  stain  the  spores  without  reference  to 
the  bodies  of  the  bacteria,  the  specimens  may,  according  to 
a  suggestion  of  Buchner,  be  placed  for  half  a  minute  in 
concentrated  sulphuric  acid.  By  this  means  the  vegetative 
forms  lose  their  power  of  staining,  whereas  the  spores, 
after  thorough  rinsing  in  water,  are  readily  susceptible  to  the 
action  of  the  carbolfuchsin„ 

Good  results  are  obtained  with  the  aid  of  the  apparently 
complicated,  but  quite  reliable,  method  of  staining  spores 
proposed  by  Moller.  The  cover-glass,  dried  in  air,  is  placed 
for  two  or  three  minutes  in  absolute  alcohol,  is  then  rinsed 
in  water,  and  kept  for  two  minutes  in  chloroform.  After 
again  rinsing  in  water  it  is  exposed  for  one  or  two  minutes 


METHODS  OF   CULTURE  AND  OF  EXAMINATION.       109 

to  the  action  of  five  per  cent,  chromic  acid,  is  again  rinsed 
in  water,  and  is  stained  for  two  minutes  in  steaming  con- 
centrated carbol-fuchsin  solution.  The  preparation  is  then 
treated  for  a  short  time  with  five  per  cent,  sulphuric  acid 
(being  passed  through  once  or  at  most  twice),  is  thoroughly 
rinsed  in  water,  is  counterstained  quite  deeply  with 
methylene-blue  or  malachite-green,  is  again  rinsed,  and  is 
dried  and  mounted  in  Canada  balsam.  The  spores  appear 
deep  red,  the  bodies  of  the  bacteria  blue  or  green. 

Staining  of  Flagella  (Loffler). — In  staining  flagella  a 
mordant  is  first  used  :  Twenty  grams  of  tannic  acid  are 
dissolved  in  80  cu.  cm.  of  hot  water,  and  50  cu.  cm.  of  a 
saturated  watery  solution  of  ferrous  sulphate  that  has  stood 
in  the  cold  for  twenty-four  hours  with  an  excess  of  ferric 
sulphate  are  added,  and  finally  10  cu.  cm.  of  a  concentrated 
alcoholic  solution  of  fuchsin.  This  fuchsin-solution  may 
be  advantageously  permitted  to  stand  exposed  to  air  for 
several  weeks  ;  it  stains  the  better  the  older  it  is. 

The  specimens  themselves  must  be  prepared  with  cover- 
slips  most  carefully  cleansed,  and  in  such  a  way  that  an 
extremely  thin  layer  is  spread  upon  the  cover-slip  with 
little  rubbing — so  much  of  the  material  as  will  adhere  to 
the  tip  of  a  platinum  needle  must  yet  be  largely  diluted. 
Quite  young  agar-cultures,  from  fourteen  to  eighteen  hours 
old,  and  at  the  most  twenty-four  hours,  are  employed,  as 
the  flagella  can  not  be  stained  in  older  cultures.  After  the 
preparations  have  been  thoroughly  dried  in  the  air,  the  mor- 
dant is  dropped  upon  them,  and  its  action  is  permitted  to 
continue  for  a  minute.  It  is  then  entirely  washed  off,  the 
cover-glass  is  dried,  and  upon  it  is  filtered  aniline-water 
gentian-violet  or  methyl-violet  or  fuchsin-solution.  Heat 
is  now  applied  carefully  by  means  of  a  small  flame,  until 
vapors  of  steam  arise.  Then,  after  a  minute,  the  prepara- 
tion is  washed  in  water,  is  dried,  and  is  mounted.  This 
modification  by  Giinther  of  Lofifier's  method  of  staining 
flagella  yields  quite  satisfactory  results.  By  another  method 
the  preparations,  dried  in  air,  are  exposed  for  from  six  to 
twelve  hours  to  the  action  of  a  two  per  cent,  solution  of  tannic 
acid  and  one-half  per  cent,  hydrochloric  acid,  are  rinsed  in 
water,  immersed  for  an  hour  in  a  watery  solution  of  iodin, 
again  washed  in  water,  and  then  stained  for  half  an  hour  in 
aniline-water  gentian-violet.  The  specimen  should  then  be 
mounted,  not  in  Canada  balsam,  but  in  solution  of  iodin. 


no  CLINICAL   BACTERIOLOGY. 


DETERMINATION   OF   THE    PATHOGENICITY   (OR   THE    SPE- 
CIFICITY) OF  BACTERIA  BY  ANIMAL  EXPERIMENTATION. 

The  possession  of  pure  cultures  renders  it  possible  to 
undertake  experiments  upon  animals,  and  thus  to  deter- 
mine the  changes  that  are  induced  in  the  animal  organism 
by  a  given  variety  of  bacteria. 

In  order  to  consider  a  bacterium  as  the  cause  of  an  in- 
fective disease,  in  order  to  declare  it  specific  for  this  disease, 
it  must  comply  with  three  conditions  formulated  by  Koch  : 
In  the  first  place,  it  must  be  present  in  all  cases  of  the  given 
disease  ;  in  the  second  place,  it  must  occur  only  in  this  dis- 
ease ;  and,  in  the  third  place,  it  must  be  capable  of  induc- 
ing essentially  the  same  disease  in  experiments  on  animals. 
Animal  experimentation,  therefore,  plays  a  most  important 
role  in  bacteriology. 

In  order  to  mfect  animals  with  bacteria  various  ways 
are  open.  The  natural  portals  of  infection  may  be  em- 
ployed, and,  besides,  new  ones  may  be  artificially  estab- 
lished through  which  the  microbes  are  introduced  into  the 
organism. 

(a)  Cutaneous  Inoculation. — Quite  superficial  wounds  of 
the  skin  are  made  in  animals  (as  in  vaccination),  and  these 
are  smeared,  by  means  of  the  platinum  wire,  with  a  small 
amount  of  pure  culture.  In  guinea-pigs  and  in  mice,  in- 
stead of  cutaneous  inoculation,  an  incision  is  made  with 
scissors  through  the  margin  of  the  ear,  and  the  injured 
places  are  rubbed  with  the  inoculating  material. 

(6)  Subcutaneous  Inoculation. — By  means  of  a  scalpel  or 
an  inoculating  needle,  a  pocket  is  formed  in  the  subcuta- 
neous connective  tissue,  into  which  the  bacterial  material  is 
introduced  ;  or  the  bacteria  suspended  in  water  or  in  bouillon 
are  introduced  beneath  the  skin  by  means  of  a  hypodermic 
syringe. 

(c)  Intravenous  Injection. — By  means  of  a  hypodermic 
syringe  the  infecting  fluid  is  injected  directly  into  a  vein 
that  either  lies  quite  superficially  (as  the  marginal  vein 
of  the  rabbit)  or  is  exposed  by  dissection.  Entrance  of 
air  into  the  vein  may  cause  immediate  death  through  air- 
embolism.  It  is  therefore  necessary  to  exclude  carefully 
all  air-bubbles  from  the  system,  and,  further,  to  compress 
the  vein  with  a  small  cotton  pad,  and  close  the  wound 
immediately  after  removal  of  the  needle. 


METHODS  OF  CULTURE  AND  OF  EXAMINATION.       Ill 

(d)  Inoculation  of  the  Anterior  Chamber  of  the  Eye. — With 
a  cataract-needle  a  small  incision  is  made  at  the  junction  of 
the  cornea  with  the  sclerotic,  the  aqueous  humor  is  per- 
mitted to  escape,  and  the  infecting  material  is  introduced. 
The  wound  closes  and  heals  rapidly. 

{e)  Inoculation  of  the  Cavities  of  the  Body. ^-The  needle 
of  the  syringe  is  introduced  into  the  selected  cavity  (pleural 
or  peritoneal),  and  the  suspension  of  bacteria  is  injected.  In 
intraperitoneal  inoculation  the  needle  of  the  syringe,  after 
thorough  cleansing  of  the  abdominal  wall,  is  introduced 
subcutaneously  in  a  horizontal  direction  ;  then  the  syringe  is 
elevated,  and  the  needle  pushed  on  until  the  disappearance 
.of  resistance  indicates  that  the  extremity  is  free  in  the 
abdominal  cavity.     . 

(/")  Subdural  Inoculation. — A  trephine-opening  is  made 
to  one  side  of  the  sagittal  suture  in  order  to  avoid  injury  to 
the  Longitudinal  sinus,  and,  with  the  aid  of  a  curved  needle, 
the  fluid  is  introduced  beneath  the  dura. 

(^g)  Inocidation  by  Inhalation. — The  bacterial  mass  is 
minutely  subdivided  by  means  of  a  spray,  which  is  intro- 
duced through  a  tube  into  a  closed  space  in  which  the 
experimental  animals  are  placed, 

(Ji)  Inoculation  through  tJie  G astro-intestinal  Tract. — The 
food  of  the  animals  is  saturated  with  the  bacterial  fluid,  or 
this  is  introduced  into  the  stomach  by  means  of  a  tube,  the 
jaws  of  the  animal  being  held  apart  with  the  aid  of  a  hollow 
wooden  gag,  through  which  an  elastic  so-called  Nelaton 
catheter  is  passed  carefully  into  the  stomach. 

For  special  purposes  (introduction  of  bacteria  into  the 
liver  or  into  the  portal  vein  or  into  a  loop  of  intestine)  celi- 
otomy becomes  necessary.  After  especially  careful  sterili- 
zation of  instruments,  hands,  and  field  of  operation,  the 
cutaneous  incision  is  made,  the  muscles  are  divided  layer  by 
layer,  and,  finally,  the  peritoneum  is  divided  upon  a  grooved 
director.  At  the  conclusion  of  the  inoculation  the  wound 
is  closed  by  interrupted  peritoneal,  muscular,  and  cutaneous 
sutures,  and  it  is  covered  with  iodoform-coUodion. 

All  of  these  various  inoculations  must  naturally  be  car- 
ried out  with  the  most  rigorous  cleanliness.  The  skin  at 
the  point  of  inoculation  must  be  shaved  and  washed  with 
soap,  solution  of  mercuric  chlorid,  alcohol,  and  ether.  All 
instruments  employed  in  the  inoculation  are  sterilized  by 
boiling  in  a  one  per  cent,  soda-solution.     The  disinfection 


112 


CLINICAL  BACTERIOLOGY. 


of  the  ordinary  hypodermic  syringe  is  more  difficult,  and 
for  this  reason  quite  a  number  of  steriHzable  syringes 
(Fig.  36)  have  been  devised  (Roux,  Koch,  Lewin).  The 
ordinary  hypodermic  syringe  is  conveniently  and  safely 
disinfected  by  filling  it  with,  and  permitting  it  to  remain 
for  from  twelve  to  twenty-four  hours  in,  a  five  per  cent, 
solution  of  carbolic  acid,  and  then  removing  the  carbolic 
acid  by  repeated  rinsing  in  sterilized  water.  In  deal- 
ing with  especially  infectious  and  conspicuously  resistant 
bacteria  (anthrax  and  tetanus),  however,  the  syringe  of 
Roux  is  employed,  and  it  is  boiled  for  ten  minutes  in  a 
one  per  cent,  solution  of  soda.  In  inoculating  the  anterior 
chamber  of  the  eye  the  greatest  care  must  be  taken  to 
secure  disinfection  of  the  conjunctival  sac.    This  is  cleansed 


Fig.  36. — I,  Roux's  bacteriologic  syringe ;  2,  Koch's  syringe ;  3,  Meyer's  bacteriologic 

syringe. 


with  a  I  :  3000  solution  of  mercuric  chlorid,  which  is 
removed  by  irrigation  with  sterilized  water.  Anesthesia  is 
effected  by  means  of  boiled  cocain-solution. 

The  inoculated  animals  are  carefully  observed,  their  tem- 
perature is  taken  at  regular  intervals,  the  evacuations  and 
the  occurrence  of  convulsions,  etc.,  are  noted,  and,  in  short, 
all  manifestations  of  disease  are  looked  for.  Should  an  in- 
oculated animal  die,  the  autopsy  is  to  be  conducted  with 
every  precaution.  It  is  usually  made  as  soon  as  possible 
after  death,  in  order  to  avoid  the  occurrence  of  putrefaction. 
The  animal,  placed  upon  its  back,  is  stretched  upon  a 
board,  with  somewhat  raised  borders.  The  whole  surface 
of  the  animal  is  moistened  with  a  solution  of  mercuric 
chlorid,  in  order  to  prevent  the  generation  of  dust.     The 


METHODS   OF  CULTURE  AND  OF  EXAMINATION.       113 

abdominal  wall  is  thoroughly  cleansed  with  the  same  solu- 
tion, and  then,  with  instruments  sterilized  in  the  flame,  it  is 
divided  and  dissected  back  on  either  side  sufficiently  for  the 
flaps  to  be  fastened  to  the  board  with  small  upholstery- 
tacks.  After  renewed  irrigation  a  fold  of  peritoneum  is 
raised,  the  abdominal  cavity  opened  with  a  freshly  sterilized 
knife,  and  the  peritoneum  thrown  back  on  either  side  as  far 
as  possible.  Pieces  of  all  the  organs  (liver,  spleen,  kidney, 
possibly  testicle)  are  removed,  and  placed  in  sterilized 
glass  jars  for  further  investigation.  From  the  tissue-fluids 
of  the  organs  and  from  the  blood  cover-slip  preparations 
are  made,  and  at  the  same  time  cultures  are  prepared  and 
poured  into  plates.  As  a  matter  of  course,  the  macroscopic 
appearance  of  the  organ  also  is  observed,  and  every  patho- 
logic alteration  is  carefully  noted.  If  necessary,  portions 
of  the  various  organs  are  hardened  and  cut  with  the  micro- 
tome, in  order  that  the  distribution  of  the  bacteria  in  the 
tissues  can  be  studied  subsequently  in  stained  sections. 

In  opening  the  thoracic  cavity  the  xiphoid  process  is 
raised  with  sterilized  forceps,  and  the  ribs  on  the  left  side, 
and  then  those  on  the  right,  are  divided  with  scissors,  and, 
finally,  the  manubrium  sterni  above  is  divided.  The  heart  is 
now  exposed,  and  is  opened  with  a  sterilized  and  cooled 
knife ;  and  cultures,  plates,  and  streak-preparations  are 
made  from  the  heart's  blood.  At  the  conclusion  .of  the 
autopsy  the  instruments  and  the  dissecting  table  are 
thoroughly  disinfected,  and  the  animal  cadaver  is  incin- 
erated. 

Special  circumstances  justify  at  times  the  holding  of  the 
autopsy  at  a  later  period,  or  subsequent  investigation  of 
individual  organs.  Thus,  typhoid-bacilli  are  more  easily 
cultivated  from  the  spleen  of  a  patient  dead  of  typhoid 
fever,  if  that  organ  has  been  kept  for  some  time,  than  if  it 
is  examined  fresh.  Also,  in  the  blood  of  rabbits  destroyed 
by  pneumonia  the  bacteria  can  be  more  readily  demon- 
strated about  twelve  hours  after  death  than  immediately 
after.  Under  these  circumstances  an  increase  of  the  bac- 
teria in  the  cadaver  must  obviously  take  place. 

However  valuable  animal  experimentation  often  proves, 
it  is,  nevertheless,  not  capable,  in  every  instance,  of  securing 
the  desired  information.  It  fails  frequently,  and  especially 
in  connection  with  those  diseases  that  occur  as  infections 
exclusively  in  man — as  for  instance,  cholera,  typhoid  fever, 
8 


114  CLINICAL  BACTERIOLOGY. 

etc.  ;  but  even  in  these  cases  experiments  on  animals 
are  not  useless,  for  they  demonstrate  the  toxic  activity 
of  the  bacteria  in  question,  and  they  lead,  above  all,  to  a 
knowledge  of  the  anti-bodies  formed  in  the  blood-serum. 
Also,  the  other  of  Koch's  postulates — that  a  microbe,  to  be 
considered  as  the  cause  of  a  disease,  must  be  present  only 
in  association  therewith — is  not  fulfilled  in  certain  diseases. 
Those  infections  that  are  due  to  the  bacteria  giving  rise  to 
inflammation  and  suppuration  are  sometimes  caused  by  the 
one  and  sometimes  by  the  other  of  these  germs.  The 
clinical  picture  of  these  diseases  depends  less  upon  the 
species  of  infecting  bacteria  than  upon  the  site  at  which  the 
infection  is  localized.  This  is  true  of  otitis  media,  of  men- 
ingitis, of  empyema,  etc.  ;  at  least,  we  are  as  yet  not  in  a 
position  with  regard  to  these  diseases  to  set  up  different 
clinical  pictures  in  accordance  with  the  various  bacterial 
findings.  A  streptococcus-meningitis  is  clinically  not 
sharply  differentiated  from  a  pneumococcus-meningitis  or 
from  a  meningitis  caused  by  staphylococci,  etc.  The  con- 
ception of  the  specificity  of  bacteria,  which  is  .  otherwise 
fully  applicable  to  the  etiology  of  infectious  diseases,  must 
be  abandoned  with  relation  to  these  common  excitants  of 
inflammation. 


PART   II 


INFLAMMATION  AND  SUPPURATION* 

Almost  all  bacteria  exhibit  under  some  circumstances  in- 
flammatory and  suppurative  properties  (phlogogenic  or  pyo- 
genic activity).  Inflammation  and  suppuration  may  also  be 
induced  by  chemic  substances — as,  for  instance,  ammonia, 
oil  of  turpentine,  etc.;  but,  above  all,  by  bacterial  metabolic 
products  (ptomains,  proteids,  etc.)  when  these  are  cm- 
ployed  apart  from  the  bacteria.  For  practical  purposes, 
however,  this  purely  chemic  mode  of  origin  is  of  no  note- 
worthy importance,  and  in  almost  every  suppurative  and 
inflammatory  process  microorganisms  are  the  responsible 
agents.  The  bacteria  that  are  found  in  the  large  majority 
of  cases  in  inflammatory  and  suppurative  areas,  and  that 
are  known  as  the  common  (not  specific)  causes  of  inflam- 
mation and  suppuration,  are  as  follows  : 

1.  The  so-called  pyogenic  cocci  (staphylococci,  strep- 
tococci, pneumococci,  etc.). 

2.  The  bacterium  coli  commune,  and  the  entire  group 
of  related  bacteria. 

3.  The  far  less  common  pneumonia-bacillus  of  Fried- 
lander. 

4.  The  bacillus  pyocyaneus. 


MORPHOLCXJY  OF  THE  CAUSATIVE  AGENTS  OF 

INFLAMMATION. 

Staphylococcus  Pyogenes  Aureus  (Fig.  37). — This  ap- 
pears in  the  form  of  spherical  nonmotile  cells  (micrococci), 
from  0.7  to  1.2  fi  in  diameter,  generally  arranged  like 
bunches  of  grapes ;  hence  the  designation  staphylococci. 
They  stain  readily  with  all  basic  aniline  dyes,  and  also 
according  to  Gram's  method.     The  temperature-minimum 

115 


116  CLINICAL  BACTERIOLOGY. 

is  +6°  C.  (42.8°  F.),  the  temperature -maximum  +44°  C. 
(111.2°  F.),  the  temperature-optimum  from  +34°  0.(93.2° 
F.)  to  -{-38°  C.  (100.4°  F-)  •  thus,  the  temperature  of  the 
body.  On  gelatin-plates,  with  low  powers  of  the  micro- 
scope, they  form  at  first  round,  coarsely  granular  colonies, 
with  sharply  limited  borders,  and  of  whitish-gray  color  ; 
later,  they  become  orange -yellow,  and  liquefy  the  gelatin 
with  moderate  rapidity.  In  gelatin  stab-cultures  develop- 
ment takes  place  along  the  line  of  inoculation,  with  liquefac- 
tion. On  agar  streak-cultures  there  forms  a  moist,  shining, 
golden-yellow  raised  column,  and  also  upon  potatoes. 
Bouillon  is  rendered  densely  turbid,  and  presents  a  yellow 
sediment.  Milk  is  coagulated.  In  milk  and  bouillon,  prin- 
cipally lactic  acid,  also  propionic  acid,  valerianic  acid,  and 


^ 


^'i 


^tt^ 


Fig,  37.— Staphylococcus  pyogenes  aureus,  from  an  agar-agar  culture  (Giinther). 

isobutyric  acid,  are  formed.  Facultative  anaerobic,  the 
staphylococcus  aureus  generates  its  yellow  pigment  only  in 
the  presence  of  oxygen.  Cultures  retain  their  vitality  for 
more  than  a  year.  They  are  destroyed  by  brief  exposure 
to  the  action  of  live  steam,  and  in  pus  dried  on  silk  threads 
by  exposure  to  the  action  of  two  or  three  per  cent,  carbolic 
acid  for  five  minutes. 

Staphylococcus  pyogenes  albus  (Fig.  38)  is  absolutely 
identical  with  the  foregoing  except  that  it  does  not  give 
rise  to  pigment-formation. 

Staphylococcus  pyogenes  citreus  generates  a  citron - 
yellow  ferment,  but  in  its  other  properties  it  is  identical 
with  the  staphylococcus  aureus. 


MORPHOLOGY  OF  CAUSATIVE  AGENTS. 


117 


Both  staphylococcus  cere  us  albus  and  staphylococcus  cereus 
flavus,  which  are  uncommon,  are  characterized  by  not 
liquefying  gelatin.  The  one  possesses  a  wax-like  white 
color,  while  the  other  generates  a  wax-like  yellow  pigment. 


Fig.  38. — Staphylococcus  pyogenes  albus  (Jakob). 

Streptococcus  Pyogenes  (Erysipelatis). — This  appears 
in  the  form  of  nonmotile  micrococci  arranged  in  chains 
of  greater  or  lesser  length.     (Fig.  39.)     They  vary  in  size 


Fig-  39- — Streptococcus  pyogenes  (Jakob). 


between  0.3  At  and  i  ai.  They  stain  readily,  and  by  Gram's 
method.  The  temperature-optimum  is  between  30°  C. 
(86°   F.)   and   37°    C.  (98.6°   F.)  ;  they  develop   also    at 


118  CLINICAL  BACTERIOLOGY. 

room-temperature,  which,  however,  must  not  be  too  low. 
Upon  gelatin-plates  the  streptococcus  grows  in  the  form  of 
white,  small,  granular  colonies  that  do  not  cause  liquefac- 
tion. With  high  powers  of  the  microscope  the  chains  can 
distinctly  be  seen  projecting  beyond  the  margin  of  the 
plate.  In  gelatin  stab-culture  there  is  no  confluent  growth  ; 
the  line  of  inoculation  can  be  seen  distinctly  to  be  made 
up  of  individual  colonies  separated  from  one  another.  The 
stab-culture  on  agar  presents  the  same  appearance. 
Bouillon,  which  constitutes  an  admirable  culture-medium 
for  streptococci,  is  usually  not  rendered  turbid,  but  it 
presents  a  flocculent  crumbling  sediment.  Upon  potatoes 
only  slight  growth  takes  place.  In  milk  the  streptococcus 
induces  coagulation.  It  exhibits  facultative  anaerobiosis. 
In' cultures  the  streptococcus  dies  much  more  quickly  than 
the  staphylococcus — within  as  short  a  time  as  four  months. 
Accordingly  as  the  streptococci  develop  in  bouillon  into  long 
or  short  chains,  a  distinction  has  been  made  between  strepto- 
coccus longiis  and  streptococcus  brevis.  Besides,  a  streptococcus 
conglomeratus  has  been  distinguished  that  resembles  super- 
ficially the  staphylococcus  in  the  interlacing  and  adhesion 
of  the  individual  chains.  This  differentiation,  however, 
has  had  to  be  abandoned.  In  order  to  maintain  the  vir- 
ulence of  streptococci,  Petruschky  recommends  their  re- 
newal in  gelatin  stab-cultures  every  five  days,  and  their 
preservation  in  the  refrigerator. 

Diplococcus  Pneumoniae  Frankel  {Streptococcus  Lanc- 
eolatus  Pasteur^. — This  organism  is,  as  a  rule,  a  nonmotile 
coccus,  arranged  in  pairs,  with  lancet-shaped  extremities,  and 
frequently  joined  together  in  small  chains.  (Figs.  40,  41.) 
They  possess  a  capsule,  which,  however,  is  only  clearly 
visible  in  the  products  of  disease,  but  which  is  usually  ab- 
sent in  cultures.  They  stain  readily,  and  also  by  Gram's 
method.  The  capsule  can  be  demonstrated  by  placing  the 
cover-glass  preparation  for  a  minute  in  i  per  cent,  acetic 
acid,  drying,  and  then  staining  in  aniline-water  gentian- 
violet.  Johne's  method  of  staining  capsules  may  also  be 
employed  (p.  108).  The  temperature-minimum  is  22°  C. 
(71.6°  F.)  ;  the  temperature-maximum  39.5°  C.  (103.1°  F.) 
for  cultures  upon  solid  media,  42.5°  C.  (108.5°  F.)  for  cul- 
tures in  fluid  media  ;  the  temperature-optimum  from  35°  C. 
(95°  F.)  to  37°  C.  (98.6°  F.).  Upon  gelatin  the  pneumo- 
coccus  appears   at  temperatures   above  25°  C.  (yj^  F.)  in 


MORPHOLOGY  OF  CAUSATIVE  AGENTS. 


119 


the  form  of  small,  delicate  colonies.  Upon  solidified  agar 
in  slants,  when  only  feebly  alkaline,  upon  agar-plates  and 
upon  blood-serum  it  develops  in  the  form   of  small,   finely 


# 


i 


Fig.  40. — Diplococcus  pneumoniae  in  the  blood  (Frankel  and  PfeifFer). 


granular  colonies,  resembling  dewdrops.  Development  in 
bouillon  presents  nothing  characteristic.  Some  varieties 
induce  coagulation  in  milk.  The  cultures  die  with  extra- 
ordinary rapidity,  usually  in  the  course  of  a  few  days.  The 
cause  of  death  has  been  attributed  to  the  formation  of  lac- 
tic acid  and  formic  acid,  which  can 
always  be  demonstrated  in  cultures 
several  days  old.  If  the  cultures 
are  neutralized  with  calcium  carbon- 
ate, they  may  retain  their  vitality 
for  several  months.  The  diplo- 
coccus of  Frankel  thrives  best 
upon  culture-media  that  contain 
considerable  blood.  This  is  added 
to  solid  media  in  a  thick  layer, 
or  it  is  mixed  in  considerable 
amounts  with  fluid  media.  The 
diplococcus  grows  well  in  the 
absence  of  oxygen  and  thus  retains  longer  its  vitality  and 
its  virulence. 

Diplobacillus  Pneumoniae  Friedlander. — Much  larger 
than  the  preceding  (minimum,  i  //),  the  bacterium  of  Fried- 


f4« 


Fig:.  41- — Diplococcus  pneu- 
moniae: a,  Cocci  without  cap- 
sules ;  b,  single  and  paired 
cocci  with  capsules ;  c,  chain- 
form  ;  d,  colony  of  cocci  (Zieg- 
ler). 


®®V  III 


120  CLINICAL  BACTERIOLOGY. 

lander  is  rod-shaped.     It  is  arranged  in  pairs  and  in  chains. 

(Fig.  42.)     It  also  possesses   a   capsule,  which  is  distinct, 

except  in  cultures.     It  does  not  stain  by  Gram's  method. 

The  bacillus  grows  vigorously  as  well  at  room-tempera- 
ture as  at  the  temperature  of  the 
body.  Upon  gelatin-plates  it  forms 
small,  porcelain-like  points.   The  gela- 

®  ^    ^  \\\/^^  I 'I       ^^^  ^^  ^^^  liquefied,  but  in  the  course 

^  x^   \»\         III       of  time  is  discolored  brown.     A  stab - 

culture  assumes  a  typical  nail-shape  ; 

an  agar  streak-culture,  a  thick  succu- 

Fig.    42. —  Bacillus  pneu-  1  1  t  ^  ^ 

moniae  of  Friediander.  lent  layer,  lu  an  agar  stab-culture 
gas  is  generated.  Potato  exhibits  a 
yellowish  coating  and  gas-formation.  Grape-sugar  nutrient 
solutions  undergo  fermentation,  with  the  formation  of  car- 
bon dioxid,  hydrogen,  ethylic  alcohol,  and  acetic  acid. 
Milk  is  not  coagulated.  Friedlander's  bacillus  retains  its 
power  of  development  for  a  long  time. 

Bacillus  Pyocyaneus. — The  bacilli  of  green  or  blue 
pus  are  small,  slender,  exceedingly  active  motile  rods  (with 
a  single  flagellum)  that  in  culture  sometimes  arrange  them- 
selves in  small  chains.  They  do  not  stain  by  Gram's 
method.  They  grow  almost  as  well  at  room-temperature 
as  in  the  thermostat.  They  exhibit  facultative  anaerobiosis, 
but  no  spore-formation.  On  gelatin-plates  flat,  irregularly 
circumscribed  colonies  form,  with  a  radiate  arrangement, 
about  which  a  zone  of  liquefaction  soon  forms.  In  gelatin 
stab-culture  liquefaction  takes  place  rapidly.  Upon  agar 
and  potatoes  there  is  vigorous  development.  Bouillon  is 
rendered  densely  turbid.  Milk  is  coagulated  and  pepton- 
ized. All  cultures,  especially,  however,  those  containing 
grape-sugar,  soon  assume  a  green  or  a  greenish-blue  color 
that  is  imparted  to  the  entire  nutrient  medium.  The  bacil- 
lus pyocyaneus  generates  various  pigments,  according  to 
the  constitution  of  the  culture-medium,  but  only  with  free 
access  of  oxygen.  The  best  known  of  these  is  pyocyanin, 
a  crystallizable  aromatic  combination,  related  to  anthracene 
(Ledderhose),  and  a  fluorescent  green  pigment. 

Bacterium  Coli  Commune. — This  appears  as  short, 
narrow  rods,  frequently  with  vacuoles,  twice  as  long  as 
wide,  arranged  in  pairs.  (Fig.  43.)  Besides  this  simple  form, 
there  is,  however,  marked  pleomorphism,  with  long  rods, 
coccus-like  bodies,  and  filaments.    Motility  is  generally  quite 


MORPHOLOGY  OF  CAUSATIVE  AGENTS. 


121 


active,  although  it  may  at  times  be  wanting  completely. 
The  motile  varieties  possess  flagella.  For  staining,  Loffler's 
methylene-blue  solution  or  carbolfuchsin  is  most  available. 
The  bacteria  do  not  stain  by  Gram's  method.     They  thrive 


Fig-  43- — Bacillus  coli  communis,  from  an  agar-agar  culture  ;  X  looo  (Itzerott  and 

Niemann). 


Fig.  44. — Bacillus  coli  communis :  superficial  colony  two  days  old  upon  a  gelatin-plate ; 

X  21  (Heim). 


best  at  body-temperature,  although  they  grow  well,  also,  at 
room-temperature.  Upon  gelatin-plates  (Fig.  44)  there 
form  deeply  situated,  yellow,  punctate  colonies.  The 
superficial  colonies  either  spread  upon  the  surface,  possess 


122  CLINICAL  BACTERIOLOGY. 

a  dense  center,  and  are  surrounded  by  a  thin,  serrated,  leaf- 
like, bluish,  iridescent  coating,  which,  with  high  powers  of 
the  microscope,  exhibits  a  delicate  linear  network-arrange- 
ment ;  or  they  present  a  sharp  boundary,  and  appear  as 
porcelain-white  points  half  the  size  of  a  pinhead.  In 
gelatin  stab-cultures  a  chain  of  small,  white,  spherical  col- 
onies forms  along  the  line  of  inoculation,  while  upon  the 
surface  in  individual  cases  a  roset  appears,  and  in  other  in- 
stances a  hemisphere.  The  gelatin  is  never  liquefied,  but 
soon  exhibits  a  dense  turbidity  and  distinct  iridescence.  In 
all  gelatin-cultures  ammonia  and  crystals  of  ammonio- 
magnesium  phosphate  (coffin-lid  shape)  are  formed.  In 
agar  streak-cultures  a  dense  yellowish  deposit  forms,  and 
upon  potatoes  a  brownish  membrane.  Bouillon  is  ren- 
dered turbid,  and  in  the  case  of  the  actively  motile  varieties 
a  coating  is  often  formed.  Distinct  gas-formation  takes 
place  in  growths  upon  all  of  the  nutrient  media  mentioned. 
It  is  favored  by  the  presence  of  reducing  substances,  and 
by  anaerobic  growth.  Addition  of  potassium  nitrite  and 
sulphuric  acid  to  peptone -bouillon  cultures  (i  cu.  cm.  of  a 
0.02  per  cent,  potassium-nitrite  solution  and  several  drops 
of  sulphuric  acid  to  lo  cu.  cm.  of  bouillon)  causes  the  de- 
velopment of  a  red  tint  (nitroso-indol  reaction).  Coagula- 
tion takes  place  in  milk.  Grape-sugar,  cane-sugar,  and 
glycerin  undergo  fermentation.  Acid  urine  is  not  altered 
by  the  growth  of  the  bacterium  coli,  while  alkaline  urine  at 
times  undergoes  ammoniacal  decomposition.  The  bacterium 
coli  commune  retains  its  vitality  for  a  long  time. 

Bacillus  Aerogenes. — This  organism  is  closely  related 
to  the  preceding.  It  appears  as  short,  nonmotile  rods, 
forming  filaments,  sometimes  with  capsules,  and  with- 
out spores.  They  do  not  stain  by  Gram's  method.  Upon 
gelatin-plates  they  form  round,  porcelain-white,  prom- 
inent colonies.  In  gelatin  stab-cultures  they  form  a  nail- 
like growth,  and  gas-formation  takes  place.  In  agar 
streak-cultures  a  white,  slimy  deposit  forms.  Upon  potato 
a  yellowish  deposit  takes  place.  Bouillon  is  rendered 
turbid  and  a  coating  forms.  Milk  is  coagulated.  Growth 
takes  place  in  urine  without  decomposition  of  urea.  In 
all  nutrient  media  energetic  gas-formation  takes  place,  and 
in  those  that  contain  sugar  acetic  and  formic  acids  are  gen- 
erated. The  bacillus  aerogenes  is  probably  identical  with 
the  organism  described  by  the   Guyon   school  as  bacillus 


PATHOGENIC  PROPERTIES  OF  CAUSATIVE  AGENTS.     123 

pyogenes,  which  plays  so  important  a  role  in  the  pathology 
of  the  genito-urinary  tract. 


THE    PATHOGENIC    PROPERTIES  OF    THE    CAUSATIVE 

AGENTS    OF   INFLAMMATION    WITH   RELATION 

TO  ANIMALS. 

The  pathogenic  activity  of  the  exciting  agents  of  inflam- 
mation and  suppuration  with  regard  to  animals  is,  on  the 
whole,  much  the  same  for  all  members  of  the  group.  In- 
troduced subcutaneously,  they  cause  local  itijlammation, 
which  manifests  itself  in  simple  swelling  and  slight  febrile 
movement.  If  the  virulence  of  the  bacteria  be  somewhat 
greater,  this  inflammation  leads  to  suppuration,  and  there 
results  a  local  abscess,  which  may  rupture  outward,  and 
undergo  cure.  Still  greater  virulence  of  the  microor- 
ganisms may  cause  the  local  imflammation  and  suppuration 
to  be  complicated  by  sepsis — that  is,  by  intoxication  with 
bacterial  poisons  generated  at  the  site  of  inflammation  and 
thence  absorbed  ;  or  it  may  lead  to  pyemia — that  is,  to 
dissemination  of  the  bacteria  through  the  lymph-channels 
and  the  blood-vessels ;  in  the  latter  instance  probably 
through  emboli  containing  bacteria — and  which  give  rise 
to  the  formation  of  multiple  purulent  foci. 

The  introduction  of  the  causative  agents  of  inflammation 
into  the  serous  cavities  leads  to  the  development  of  serous 
or  purulent  pleuritis  or  peritonitis.  Under  all  of  these 
conditions  both  the  clinical  course  of  the  disease  and  the 
postmortem  findings  exhibit  completely  the  conditions 
sufficiently  well  known  from  human  pathology. 

Septicemia  is  exceedingly  common  in  animal  pathology, 
while  it  occurs  but  seldom  in  human  beings.  This  may  be 
considered  as  the  expression  of  especially  virulent  activity 
on  the  part  of  the  causative  factors  of  inflammation.  It 
occurs  after  intravenous  introduction  of  the  bacteria  ;  in 
some  cases,  however,  also  in  connection  with  every  other 
variety  of  inoculation  —  and  especially  the  infection  of 
rabbits  with  pneumococci.  Characteristic  of  septicemia  is 
the  absence  of  any  localization  of  the  disease.  The  clinical 
picture  has,  therefore,  no  distinctive  features.  The  animal 
is  severely  ill,  as  may  be  recognized  from  the  appearance 
of  the  skin,  of  the  eyes,  and  the  general  attitude.  It  does 
not  eat,  and  it  often   suffers  from  diarrhea,  especially  after 


124  CLINICAL  BACTERIOLOGY. 

infection  with  bacterium  coli  or  with  another  member  of 
this  group.  At  the  same  time  there  is  always  fever,  which 
gradually  increases  ;  and  usually  also  dyspnea.  After  the 
disease  has  existed  for  a  shorter  or  a  longer  period  of  time, 
death  results,  with  decline  of  temperature,  and  not  rarely 
with  convulsions.  Upon  postmortem  examination  the 
individual  organs  exhibit  parenchymatous  cloudiness,  the 
spleen  is  enlarged,  there  is  often  nephritis,  but  there  are 
otherwise  no  special  alterations.  On  microscopic  exam- 
ination and  cultural  investigation,  however,  the  bacteria 
introduced  can  be  seen  in  large  numbers  everywhere  :  in 
eVery  tissue,  in  the  secretions,  in  the  blood.  They  have 
multiplied  in  the  blood,  and  fill  all  of  the  vessels  down  to 
the  smallest  capillaries,  which,  by  reason  of  their  numbers, 
they  often  actually  plug.  The  bacteria  are  demonstrable 
in  the  blood  already  during  life,  after  the  disease  has 
existed  for  some  time,  but  generally  not  in  the  numbers 
in  which  they  are  found  after  death.  Their  greatest  multi- 
plication appears  to  take  place  just  before  death,  and  in 
the  cadaver. 

The  pathogenic  activity  of  the  individual  causative  agents 
of  inflammation  may  be  stated  as  follows  : 

Staphylococci :  Cutaneous  inoculation  is  unattended 
with  ill  results  ;  subcutaneous  injection  induces  a  local 
abscess  in  mice,  guinea-pigs,  rabbits  ;  and  intravenous  injec- 
tion, at  times,  pyemia  in  rabbits. 

Streptococci :  If  the  material  is  rubbed  upon  small 
cutaneous  wounds  of  the  rabbit's  ear,  erysipelatous  inflam- 
mation follows.  Subcutaneous  injections  in  mice  and 
guinea-pigs  are  followed  by  septicemia,  with  or  without 
local  abscess  ;  and  intravenous  injection  induces  septicemia. 

Diplococcus  lanceolatus  Frankel  is  pathogenic  for  rab- 
bits, mice,  and  guinea-pigs.  Death  from  septicemia  occurs 
with  certainty  in  rabbits  after  subcutaneous  injection  of  even 
small  amounts.  Mice  are  somewhat  less  susceptible,  but  they 
die  mostly  of  pneumococcus-septicemia  ;  and  guinea-pigs, 
only  after  the  introduction  of  considerable  amounts.  After 
intraperitoneal  injection  guinea-pigs  frequently  die  in  con- 
sequence of  fibrinous  peritonitis.  In  dogs  the  inflammation 
caused  by  the  diplococcus  remains  local,  the  bacteria  not 
entering  the  blood ;  the  dogs,  however,  die  as  a  result  of 
the  poisoning  (sepsis),  and  most  readily  after  subcutaneous 
inoculation. 


PATHOGENIC  PROPERTIES  OF  CAUSATIVE  AGENTS.     125 

Diplobacillus  pneumoniae  Friedlander  readily  destroys 
mice  and  guinea-pigs  on  subcutaneous  and  intravenous  in- 
jection, and  dogs  and  rabbits  less  readily.  Postmortem 
examination  discloses  here,  also,  septicemia. 

Bacillus  pyocyaneus,  when  employed  in  considerable 
amounts,  is  pathogenic  for  guinea-pigs  and  rabbits,  inducing 
local  suppuration,  diarrhea,  and  sepsis. 

Bacterium  coli  commune :  Mice,  guinea-pigs,  rabbits, 
and  dogs  are  susceptible  to  subcutaneous,  intraperitoneal 
or  intravenous  injection.  There  result  local  abscesses, 
hemorrhagic  diarrhea,  and  septic  manifestations ;  post- 
mortem examination  disclosing  marked  hyperemia  of  all 
the  abdominal  organs,  and  often  septicemia. 

In  conclusion,  it  may  be  again  especially  emphasized  that 
the  results  obtained  in  experiments  on  animals  with  all  of 
these  common  causative  agents  of  inflammation  are  by  no 
means  constant.  They  vary  quite  extraordinarily,  in  ac- 
cordance with  the  virulence  of  the  material  employed,  and 
the  inoculations  frequently  enough  pursue  a  completely 
negative  course.  The  virulence  of  the  exciting  agents  of 
inflammation  varies,  however,  according  to  their  source. 
The  bacteria,  for  instance,  cultivated  from  a  fatal  pyemia, 
are  much  more  virulent  than  the  relatively  harmless  bacte- 
ria derived  from  a  mild  suppuration  of  the  skin,  etc.  It  is 
to  be  emphasized,  further,  that  all  of  these  exciting  agents 
of  suppuration  and  inflammation  are  not  exclusively 
pyogenic,  but  they  may  also  induce  purely  serous  pro- 
cesses :  as,  in  the  main,  the  three  principal  varieties  of  in- 
flammation— the  serous,  the  fibrinous,  and  the  purulent — 
differ  from  one  another  only  quantitatively. 


THE    OCCURRENCE    OF    THE    CAUSATIVE   AGENTS    OF 

INFLAMMATION  AND  SUPPURATION  IN  HEALTHY 

PERSONS  AND  OUTSIDE  THE  BODY. 

Staphylococci  have  been  found  in  dust,  upon  the  sur- 
face of  the  earth,  in  the  air,  in  household  wash-water  ; 
further,  almost  constantly  upon  the  surface  of  the  skin,  in 
the  accumulations  beneath  the  finger-nails,  in  the  saliva, 
in  the  mucus  of  the  pharynx,  in  the  nasal  secretion,  in 
the  intestinal  contents,  in  the  vagina,  and  in  the  urethra. 

Streptococci  have  been  cultivated  from  the  air  of  hospi- 


126  CLINICAL  BACTERIOLOGY. 

tal-wards  and  dissecting  rooms,  from  the  mouth,  the  nose, 
the  pharyilx,  and  the  skin  ;  further,  from  the  intestine, 
and — though  selfiom — from  the  vagina  of  healthy  women. 

The  diplococcus  pneumoniae  Frankel  is  an  exceed- 
ingly frequent  inhabitant  of  the  buccal  and  nasal  cavities, 
and  especially  of  the  entire  upper  portion  of  the  respira- 
tory apparatus  ;  at  times  it  is  present  in  the  intestine. 

The  diplobacillus  Friedlander  is  found  normally  in  the 
same  situations  as  the  diplococcus  Frankel,'  although  its 
occurrence  is  much  more  seldom. 

The  bacillus  pyocyaneus  has  been  found  upon  the 
skin,  especially  in  the  axillary  cavity  ;  further,  in  the 
external  auditory  canal  and  in  intestinal  mucus  ;  it  is  not 
rarely  encountered  also  in  the  air  and  in  water. 

The  bacterium  coli  commune  and  bacterium  lactis 
aerogenes  are  found  throughout  the  entire  digestive  tract, 
especially  in  the  intestine  (the  aerogenes  more  commonly  in 
the  feces  of  infants) ;  further,  in  the  vulva  and  the  vagina 
and  on  the  prepuce,  upon  the  skin,  in  the  air,  in  water,  and 
in  milk. 

The  common  causative  agents  of  inflammation  and 
suppuration  are  thus  found  in  healthy  persons  in  the  main 
upon  the  skin  and  the  so-called  open  cavities  of  the  body, 
the  pyogenic  cocci  more  commonly  upon  the  surface  of  the 
skin  and  in  the  upper  portion  of  ^the  digestive  and  respira- 
tory tracts,  and  the  pyogenic  bacterium  coli  more  com- 
monly in  the  intestine.  It  is,  therefore,  to  be  anticipated 
that  the  inflammatory  and  suppurative  processes  that  take 
place  upon  the  skin  and  in  the  neighborhood  of  the  buccal, 
nasal,  and  pharyngeal  cavities  are  excited  especially  by 
cocci,  while  those  of  the  intestinal  tract  and  of  the  genito- 
urinary tract  and  their  entire  neighborhood  are  due  more 
commonly  to  the  bacterium  coli  or  the  bacterium  lactis 
aerogenes. 


THE   OCCURRENCE    OF    THE    CAUSATIVE   AGENTS  OF 
INFLAMMATION  IN  DISEASE. 

CUTANEOUS    SUPPURATION. 

Furuncle  and  carbuncle  are  almost  always  attended 
with  the  presence  of  staphylococci  (staphylococcus  aureus 
and  albus).     That  staphylococci  are,  in  fact,  the  cause  of 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  127 

carbuncles  has  been  demonstrated  by  Garre,  in  an  experi- 
ment upon  himself.  He  rubbed  a  staphylococcus-culture 
into  the  intact  skin  of  his  left  forearm.  Four  days  later  a 
characteristic  carbuncle  developed,  and  around  it  several 
isolated  furuncles.  The  pus  from  all  of  the  lesions  con- 
tained the  same  staphylococcus  that  had  been  employed 
for  the  inoculation.  This  experiment  renders  it  at  the 
same  time  probable  that  furuncles  and  carbuncles  are 
probably  due  to  infection  of  the  excretory  ducts  of  the 
glands  of  the  skin,  into  which  the  pyogenic  material  is  in 
some  way  forced.  Practically,  furuncles  are  often  seen  to 
develop  in  places  subjected  to  pressure  or  friction  :  as,  for 
instance,  by  parts  of  the  clothing. 

Panaris. — In  the  pus  from  panaris  staphylococci,  as  well 
as  streptococci,  and  in  rare  cases  also  the  bacterium  coli  com- 
mune have  been  found. 

Abscess  and  Phlegmon. — These  are  due  to  staphylo- 
cocci and  streptococci,  and  in  some  cases  also  to  Frankel's 
pneumococci,  especially  in  childhood  and  in  the  course  of 
genuine  croupous  pneumonia.  In  abscesses  in  those  sick 
with  and  convalescent  from  typhoid  fever,  staphylococci 
and  streptococci  are  found  frequently,  and  not  at  all  seldom 
also  typhoid-bacilli  and  bacterium  coli.  Urinary  pJilegmons, 
so  frequently  observed  in  the  sequence  of  urinary  infiltra- 
tion, are  usually  due  to  the  bacterium  lactis  aerogenes  or  the 
bacterium  coli  commune,  which  play  the  most  important  role 
in  the  suppurative  processes  of  the  genito-urinary  apparatus. 

So-called  cold  abscesses  are  generally  found  to  be  sterile. 
They  may  be  considered  as  the  product  of  tubercle-bacilli, 
and  actually  it  has  frequently  been  possible  in  experiments 
on  animals  to  induce  tuberculosis  with  the  pus  from  such 
abscesses.  Microscopically,  however,  it  has  been  possible, 
only  in  the  rarest  of  instances,  to  demonstrate  in  the  pus 
the  tubercle-bacilli  that  are  present  in  small  number.  Also, 
in  the  larger,  nontuberculous  abscesses  bacteria  are  some- 
times not  found  at  the  center  of  the  area  of  suppuration, 
and  the  pus,  therefore,  appears  sterile.  It  is  only  necessary 
in  such  cases  to  examine  material  from  the  periphery,  the 
so-called  abscess-membrane,  in  order  to  find  the  pyogenic 
agents  without  difficulty. 

Gas-abscesses,  like  ordinary  abscesses,  do  not  possess 
a  uniform  etiology.  From  them  there  have  thus  far  been 
cultivated  :  (i)  The  bacterium  coli  commune  and  the  bac- 


128  CLINICAL   BACTERIOLOGY. 

terium  lactis  aerogenes,  principally  from  the  gas-phlegmons 
in  the  neighborhood  of  the  intestinal  canal  ;  (2)  a  special 
bacillus — the  bacillus  emphysematosus.  (Fig.  45.)  This 
appears  in  the  form  of  nonmotile,  plump  rods,  forming  fila- 
ments. These  are  anaerobic,  and  they  stain  by  Gram's 
method.  They  grow  slightly  in  gelatin,  without  causing 
liquefaction.  In  bouillon,  and  also  in  agar,  they  give  rise  to 
the  formation  of  fetid  gas.  This  bacillus  is  but  seldom  found 
alone  in  gas-abscesses,  but  almost  always  in  association  with 
the  ordinary  pyogenic  cocci.  In  experiments  on  animals  a 
severe  nonsuppurative  inflammation,  with  gas-formation,  and 
which  at  times  causes  death,  is  induced  in  guinea-pigs  by  sub- 


Fig.  45. — Bacillus  aerogenes  capsulatus  (from  photograph  by  Prof.  Simon  Flexner). 


cutaneous  injection  of  this  bacterium.  That  the  bacterium 
coli  and  the  bacterium  lactis  aerogenes  may  at  times  cause 
gas-phlegmons  is  not  surprising,  as  it  is  known  that  both  are 
active  gas-producers,  especially  in  the  absence  of  oxygen. 

In  the  bacteriologic  investigation  of  a  gas -abscess  it  is 
always  necessary  to  be  prepared  for  the  presence  of  anae- 
robic microorganisms,  and  the  choice  of  a  culture -method 
must  be  governed  with  reference  thereto. 

Impetigo. — This  is  a  peculiar  anatomic  form  of  suppura- 
tion within  the  layers  of  the  epidermis  that  leads  to  the 
formation  of  pustules.  From  the  contents  of  the  pustules 
the    staphylococcus   pyogenes   albus   and   aureus   and  the 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  129 

cereus  albus  are  obtained.  These  observations  with  ree^ard 
to  the  common  exciting  agents  of  inflammation  explain  the 
cHnical  fact  that  the  most  variable  suppurative  processes — 
furunculosis,  etc. — sometimes  precede  and  sometimes  follow 
the  actual  attack  of  impetigo. 

Ecthyma. — This  also  is  a  form  of  cutaneous  suppura- 
tion in  which  the  pyogenic  cocci  are  found.  From  the  pus 
staphylococci  and  streptococci  have  been  cultivated. 

Herpes. — Herpes  zoster  has  been  designated  by  Pfeiffer 
(Weimar)  an  infectious  disease  whose  causative  agent 
belongs  to  the  class  of  protozoa.  The  cells  believed  by 
Pfeiffer  to  be  protozoa  have,  however,  not  received  recog- 
nition, and,  above  all  things,  it  has  as  yet  been  impossible 
to  cultivate  them.  In  the  vesicles  that  become  turbid 
staphylococci  and  streptococci  are  always  found,  while  the 
contents  of  the  clear  vesicles  are  often  sterile. 

In  cases  of  herpes  labialis  the  vesicles  contain  the  excit- 
ing agents  of  inflammation  from  the  beginning,  and  more 
frequently  streptococci  and  Frankel's  diplococci  than 
staphylococci.  According  to  Pfeiffer,  protozoa  are  absent 
in  this  form  of  herpes.  As  soon  as  the  contents  of  the 
vesicles  are  turbid,  staphylococci  will  be  found  present 
therein,  and  partly  in  association  with  streptococci,  partly 
alone.  These  observations  suggest  that  herpes  labialis  is 
not  a  true  zoster.  In  general  it  occurs  only  as  a  complica- 
tion of  such  infectious  diseases  as  themselves  stand  in  etio- 
logic  relation  with  the  common  exciting  agents  of  inflam- 
mation (pneumonia,  meningitis,  etc.) ;  it  may,  perhaps,  be 
viewed  as  a  secondary  localization  of  the  causative  agent 
of  the  primary  process. 

In  herpes  of  the  pharynx  (angina  herpetica)  and  of  the 
larynx  the  same  conditions  seem  to  prevail  as  in  herpes 
labialis. 

ERYSIPELAS. 

Erysipelas  is  excited  by  the  streptococcus.  The  old 
discussion  whether  or  not  the  streptococcus  erysipelatis 
(Fehleisen)  is  distinct  from  the  streptococcus  pyogenes,  the 
exciting  agent  of  suppuration,  can  now  be  considered  as 
finally  decided.  The  two  microorganisms  are  without  doubt 
identical.  This  is  demonstrated  as  well  by  their  complete 
agreement  in  morphologic  and  cultural  peculiarities,  as  also 
by  the  results  of  experiments  on  animals  and  man. 
9 


130  CLINICAL  BACTERIOLOGY. 

Microscopic  Arrangement  of  the  Streptococci  in  the 
Invaded  Skin. — As  suggested  by  Fehleisen,  three  zones 
may  be  distinguished  :  (i)  A  central,  in  which  the  process 
is  in  retrogression  ;  (2)  the  elevated  erysipelatous  margin  ; 
and  (3)  a  peripheral  zone  surrounding  this  deeply  red  ery- 
sipelatous zone,  which,  microscopically,  is  apparently  still 
completely  normal.  Streptococci  are  found  in  each  of 
these  three  zones,  but  in  much  the  largest  number  in  the 
erysipelatous  margin  and  in  smaller  number  in  the  central 
and  in   the   peripheral  zone.       The  organisms   lie  in  the 


Fig.  46 — Streptococcus  erysipelatis,  seen  in  a  section  through  human  skin  ;  X  500 
(Frankeland  Pfeiffer). 


lymph-spaces  and  the  lymph-vessels  of  the  skin  and  the 
subcutaneous  fatty  tissue ;  the  lymph-spaces  are  almost 
completely  choked  up  by  them.  Sometimes  they  collect 
also  about  the  lymph-vessels  and  the  blood-vessels.  In 
the  vesicles  in  cases  of  erysipelas  bullosum  streptococci 
are  not  constantly  present.  On  the  other  hand,  they  are 
never  absent  from  the  purulent  products  oi phlegmonous  ery- 
sipelas. As  a  rule,  in  cases  of  erysipelas  pursuing  a  favor- 
able course  the  streptococci  do  not  gain  entrance  into  the 
blood.     Metastases  are,  therefore,  rare    in   the  symptom- 


OCCURRENCE  OF  CAUSATIVE   AGENTS.  131 

complex  of  erysipelas.  Cases  are,  however,  on  record  in 
which  it  was  possible  to  cultivate  streptococci  from  the 
blood  of  patients  ;  general  infection,  therefore,  having  existed. 

Immunity. — Recovery  from  one  attack  of  erysipelas 
confers  no  immunity  to  subsequent  attack.  Erysipelas  is 
one  of  the  diseases  attended  with  numerous  recurrences. 
The  blood-serum  of  persons  just  recovered  from  an  attack 
of  erysipelas  does,  however,  at  times  possess  immunizing 
properties  ;  and  large  amounts  thereof  may,  according  to 
some  reports,  immunize  animals  (guinea-pigs  and  mice) 
to  streptococci. 

Experimental  Evidence  of  the  Etiologic  Significance 
of  the  Streptococcus  for  the  Development  of  Erysipelas. 
— The  introduction  of  streptococcus-material  into  shallow, 
superficial  wounds  of  rabbits'  ears  is  followed  by  the  devel- 
opment of  erysipelatous  inflammation.  Much  more  im- 
portant are  the  observations  that  have  been  made  in  human 
beings.  Proceeding  from  the  clinical  experience  that  in 
individuals  with  advanced  lupus  or  with  inoperable  tumors 
an  attack  of  intercurrent  erysipelas  may  lead  to  the  cessa- 
tion, or  even  the  cure,  of  the  original  disease-process,  it 
was  ventured  in  such  cases  to  undertake  cutaneous  inocu- 
lations with  streptococcus-cultures  derived  from  cases  of 
erysipelas.  These  experiments  yielded  positive  results  : 
typical  erysipelas  pursuing  a  characteristic  course  occurring 
almost  constantly.  A  similar  result  was  attained  by  Koch 
and  Petruschky  by  means  of  a  streptococcus  derived  from 
a  purely  suppurative  process.  There  is  no  doubt  that  a 
therapeutic  influence  is  exerted  by  several  streptococcus- 
infections  upon  the  course  of  carcinoma,  but  the  strength 
of  the  patient  suffers  so  much  in  consequence  of  the  inocu- 
lations with  erysipelas  that  a  practical  application  of  this 
method  is  entirely  out  of  consideration. 

The  Occurrence  of  Other  Microorganisms  in  Erysipe- 
las.— In  two  cases  of  true  erysipelas  (the  second  infected 
by  the  first)  Jordan  cultivated. the  staphylococcus  pyogenes 
aureus.  From  this  it  may  be  concluded  that  the  other 
causative  agents  of  inflammation  are  capable,  under  certain 
circumstances,  of  inducing  erysipelas.  The  usual  cause, 
however,  is  without  question  the  streptococcus. 

The  hacteriologic  diagnosis  of  erysipelas  is  only  most 
rarely  necessary.  In  such  an  event  a  small  scarification  is 
made  with  a  sterilized  lancet  at  the  border  of  the  inflamma- 


132  CLINICAL  BACTERIOLOGY. 

tory  area,  and  plates  are  prepared  from  the  fluid  that 
escapes  ;  or  four  or  five  obliquely  solidified  gelatin-tubes  or 
agar-tubes  are  smeared  therewith.  In  this  way,  in  most  cases, 
individual  colonies  of  streptococci  will  be  obtained  probably 
in  the  first  tube,  and  with  certainty  in  the  remaining  tubes. 
Specific  Treatment  of  Erysipelas  and  of  Streptococ- 
cus-diseases Generally. — Marmorek  increased  the  viru- 
lence of  streptococci,  which  are  readily  attenuated  in  artificial 
culture,  by  cultivating  them  in  bouillon  to  which  sterile 
horse -serum  had  been  added.  With  progressively  increas- 
ing doses  of  the  highly  virulent  cultures  thus  obtained  he 
immunized  horses,  whose  serum  was  then  reported  to  exhibit 
immunizing  and  curative  properties  with  relation  to  all  strep- 
tococcus-infections also  in  human  beings.  Petruschky,  who 
repeated  the  experiments  of  Marmorek,  was,  however,  unable 
to  confirm  his  conclusions.  The  serum  of  Marmorek 
obtained  from  the  Pasteur  Institute  possessed  neither  im- 
munizing nor  therapeutic  activity,  also  in  observations  made 
upon  human  beings.  The  streptococcus  of  Marmorek  was 
most  highly  virulent  for  rabbits,  but  not  for  human  beings. 


p:hlebitis  and  lymphangitis. 

Lymphangitis,  which  arises  from  some  peripheral  in- 
flammation or  suppuration — in  some  cases  not  demonstra- 
ble— is  attended  with  the  presence  of  the  exciting  agents 
just  described,  the  pyogenic  cocci,  and  at  times  with  that 
of  the  bacterium  coli.  Also,  the  infectious  form  of  phlebitis, 
which  occurs  as  an  associated  manifestation  of  numerous  in- 
fectious diseases,  may  be  caused  by  the  same  microorganisms 
as  give  rise  to  the  original  disease.  Thus,  for  instance,  tuber- 
cle-bacilli have  been  found  in  the  phlebitides  of  tuberculous 
individuals,  and  the  streptococcus  pyogenes  in  the  phleg- 
masia alba  dolens  of  puerperal  women.  More  frequently, 
however,  the  inflammation  of  the  vein  is  the  expression  of 
a  secondary  infection,  and,  therefore,  its  causes  are,  as  a 
rule,  found  to  be  the  common  pyogenic  cocci.  The  bac- 
terial inflammation  of  the  vein  or  the  lymphatics  leads  to 
the  formation  of  a  bacteria-containing  thrombus.  This 
may  be  conceived  as  originating  from  adhesion  of  the  micro- 
organisms circulating  in  the  vessel  to  a  projecting  point  of 
its  wall,  or  of  a  valve,  or  from  the  penetration  by  the  bacteria 
of  the  wall  of  the  vessel  from  without  through  the  interme- 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  133 

diation  of  the  vasa  vasorum.  In  the  bacteriologic  examina- 
tion of  the  thrombus  it  is  important  to  know  that  the  bac- 
teria are  to  be  found  only  in  the  oldest,  first-formed  portion 
thereof,  and  on  the  corresponding  portion  of  the  wall  of  the 
vessel.  The  central  and  peripheral  portions  of  the  thrombus 
that  subsequently  form  mechanically  are  often  sterile. 


INFLAMMATIONS  OF   THE  NOSE  AND  THROAT. 

In  the  secretions  in  cases  of  acute  rhinitis,  pharyn- 
gitis, and  laryngitis,  staphylococci  and  streptococci  have 
been  found,  and  also  pneumococci  and  pneumobacilli,  the 
last  most  frequently  in  the  nose. 

In  cases  of  fibrinous  rhinitis  diphtheria-bacilli  have  been 
found  repeatedly.  In  other  cases  their  demonstration  was 
not  successful,  so  that  it  is  not  certain  whether  all  cases  of 
croupous  rhinitis  are  to  be  considered  as  instances  of  diph- 
theria. 

Ozena. — From  the  nasal  crusts  in  cases  of  ozena  a  micro- 
organism has  been  cultivated  by  various  observers  (Lowen- 
berg,  Abel,  Paulsen),  which  presents  so  close  a  resem- 
blance to  the  bacillus  pneumoniae  Friedlander  that  a  detailed 
description  will  not  be  necessary.  The  cultures  are  slimy 
and  viscid,  form  comparatively  little  gas,  and  do  not  cause 
coagulation  in  milk.  That  this  so-called  ozena-bacillus  is 
the  cause  of  the  disease  is  scarcely  probable.  The  char- 
acteristic odor  of  ozena  is  wanting  in  all  of  the  cultures. 
Further,  the  bacillus  is  never  found  in  the  diseased  struc- 
tures of  the  nose,  but  only  in  the  secretions  whose  peculiar 
putrid  decomposition  it  may  possibly  cause.  For  the 
present,  the  ozena-bacillus  may  be  considered  a  variety  of 
Friedlander's  bacillus,  slightly  modified  in  consequence  of 
the  peculiar  conditions  related  to  the  ozenous  nose. 

Rhinoscleroma. — Also  the  bacilli  found  in  rhinosclero- 
matous  tumors  resemble  Friedlander's  bacilli.  They  in- 
duce fermentation  of  sugar  and  of  milk,  though  in  slighter 
degree  than  the  pneumobacillus.  On  potatoes  an  often 
invisible,  at  times  brown,  gas-forming  coating  occurs.  In 
the  tissues  the  bacilli  are  found  almost  exclusively  in  the 
cells,  in  which  they  displace  the  nucleus  and  the  proto- 
plasm to  one  side,  themselves  almost  entirely  filling  the 
whole  cell  (Mikulicz). 

Noma. — Schimmelbusch  described  special  noma-bacilli. 


134  CLINICAL  BACTERIOLOGY. 

In  a  case  of  noma  studied  personally  the  bacterium  coli 
commune  was  found.  Concerning  these  observations  the 
same  criticism  may  be  made  that  has  been  suggested  with 
regard  to  the  presence  of  Friedlander's  bacillus  in  cases 
of  ozena  and  rhinoscleroma. 


ANGINA. 

In  the  inflamed  mucous  membrane  of  anginas  pursuing 
their  course  without  the  development  of  membrane  staphy- 
lococci have,  as  a  rule,  been  found,  as  well  as  in  the  purulent 
plugs  of  follicular  (or  lacunar)  angina.  The  anginas  char- 
acterized by  the  presence  of  a  membranous  deposit  are 
frequently  associated  with  streptococci,  and  often  besides 
also  with  staphylococci,  but  at  times  with  the  latter  alone. 
Finally,  a  considerable  number  of  cases  have  been  reported 
in  which  only  pneumococci  were  present. 

An  attempt  has  been  made  on  the  basis  of  these  bacterio- 
logic  observations  to  distinguish  a  staphylococcus-angina,  a 
streptococcus-angina^  and  a  pneumococcus-angina,  each  of 
which  is  held  to  be  etiologically  a  distinct  variety  and  to 
possess  its  special  characteristics  both  clinically  and  from 
the  diagnostic  and  prognostic  point  of  view.  Accord- 
ingly, staphylococcus-angina  would  be  a  relatively  harmless 
affection,  scarcely  ever  leading  to  complicating  disorders. 
Streptococcus-angina  would  be  severer,  exhibiting  more 
pronounced  manifestations  of  intoxication  (fever,  glandular 
enlargement),  and  possibly  being  followed  by  nephritis  and 
even  general  sepsis.  Pneumococcus-angina,  finally,  would 
be  characterized  by  the  resemblance  of  its  clinical  course  to 
that  of  pneumonia,  by  its  stormy  onset,  possibly  with  a 
chill,  and  by  high  fever,  with  critical  defervescence. 

It  must,  however,  be  emphasized  that  the  clinical  picture 
of  these  anginas  is  by  no  means  a  constant  one,  and  that 
the  severity  of  the  disease  depends  more  upon  the  virulence 
than  upon  the  species  of  the  bacteria  present.  There  occur 
staphylococcus-anginas  i^phlegmonoiis  anginas),  as  well  as 
anginas  associated  with  the  presence  of  streptococci  alone, 
that  pursue  as  mild  a  course  as  those  with  which  the  staphy- 
lococcus alone  is  associated.  On  the  other  hand,  severe 
pseudo-membranous  anginas,  which  clinically  are  indistin- 
guishable from  true  diphtheria,  and  in  association  with 
which   only  streptococci   are   demonstrable,   are   not   rare. 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  135 

Pneumococcus-angina,  further,  pursues  frequently  the  char- 
acteristic course  outlined,  though  by  no  means  always. 
It  is  to  be  added  that  isolated  cases  of  tonsillitis  have  been 
reported  in  which  the  bacterium  coli  commune  exclusively 
has  been  found. 

Above  all,  however,  in  a  not  inconsiderable  number  of 
cases  of  angina,  there  is  mixed  infection  with  several  of  the 
micrococci  named.  Then,  most  commonly  the  two  varieties 
of  bacteria  are  found  together  from  the  beginning.  At 
times,  however,  only  streptococci  can  be  demonstrated  at 
first,  and  after  some  days  also  staphylococci,  or  even  the 
latter  alone.  Under  these  conditions  it  must  be  concluded 
that  the  one  coccus  has  associated  itself  with  or  overgrown 
the  other.  The  frequency  of  these  mixed  infections,  which 
clinically  are  indistinguishable  from  the  pure  infection,  alone 
renders  impossible  a  rigid  separation  of  the  anginas  accord- 
ing to  their  causative  agents. 

Scarlatinal  angina  is  usually  associated  with  the  presence 
of  streptococci. 

The  bacteriologic  diagnosis  can,  according  to  what  has 
been  said,  be  made  only  with  a  certain  degree  of  probability 
from  the  clinical  picture  and  the  course  of  the  disease.  A 
positive  diagnosis  is  possible  only  on  microscopic  and  cul- 
tural investigation.  The  latter  is  made  simply  by  rubbing 
a  sterile  swab  of  cotton  that  has  been  applied  to  the  tonsil, 
or  a  bit  of  secretion  or  membrane  obtained  by  means  of 
a  sterilized  platinum  loop,  upon  from  three  to  five  glycerin- 
agar  tubes,  or,  better — in  the  differential  diagnosis  of  diph- 
theria— upon  from  three  to  five  tubes  of  Lofifler's  serum,  or 
upon  a  serum-plate.  The  bacterial  decision  will  frequently 
be  questionable  on  account  of  the  impossibility  of  exclud- 
ing diphtheria  with  certainty  by  other  means.  Detailed 
reference  will  be  made  to  this  point  in  the  section  on 
Diphtheria. 

From  what  has  been  said,  it  will  be  clear  that  the  dem- 
onstration of  staphylococci,  streptococci,  or  pneumococci,  is 
available  for  purposes  of  prognosis  only  with  great  caution. 


OTITIS  MEDIA. 
In  cases  of  serous  inflammation  of  the  middle  ear,  as 
well  as  in  cases  of  suppurative  or  hemorrhagic  type,  there 
have  been  found  the  diplococcus  pneumoniae  Frankel,  the 


136  CLINICAL  BACTERIOLOGY. 

streptococcus  pyogenes,  the  staphylococcus  pyogenes,  the 
diplobacillus  pneumoniae  Friedlander,  the  bacillus  pyocya- 
neus,  alone  or  in  mixed  infection.  All  of  these  organ- 
isms are  more  or  less  common  inhabitants  of  the  buccal 
cavity,  and  migrate  through  the  Eustachian  tube  into  the 
middle  ear  under  special  circumstances.  Influenza-otitis  is 
associated  with  the  presence  of  influenza-bacilli ;  tubercu- 
lous otitis,  constantly  with  that  of  the  tubercle-bacillus. 

Bacteriologic  Diagnosis. — The  auditory  canal  is  cleansed 
carefully  with  mercuric-chlorid  solution,  paracentesis  is 
practised  with  a  suitable  needle  or  knife  sterilized  in  the 
flame,  the  escaping  pus  is  taken  up  with  a  platinum  wire 
bent  at  an  angle,  and  from  it  plates  are  cast  (agar,  on 
account  of  the  presence  of  pneumococci). 

After  the  occurrence  spontaneously  of  perforation,  the 
examination  is  of  little  value,  as  the  secretion  will  have 
become  contaminated  by  the  numerous  microorganisms  of 
the  external  auditory  canal.  If  tuberculosis  is  suspected  in 
a  case  of  chronic  otitis  media,  the  pus  should  be  examined 
microscopically  for  tubercle-bacilli,  and  possibly  animals 
should  be  inoculated  with  it. 


MENINGITIS. 

Inflammation  of  the  cerebral  meninges  occurs,  as  a 
primary  disorder  in  the  form  of  epidemic  cerebrospinal 
meningitis,  and  as  a  secondary  (metastatic)  condition  in  con- 
nection with  pyemia  and  numerous  infectious  diseases,  espe- 
cially in  the  sequence  of  otitis  media,  inflammation  of  the 
accessory  cavities  of  the  nose,  and  croupous  pneumonia. 
A  special  position  is  occupied  by  tuberculous  meningitis, 
which  preferably  involves  the  base  of  the  brain.  From  the 
exudate  in  cases  of  secondary  meningitis  there  have  been 
cultivated  the  diplococcus  lanceolatus  Frankel,  the  strep- 
tococcus pyogenes,  the  staphylococcus  pyogenes,  the  bac- 
terium coli,  and  the  diplobacillus  pneumoniae  Friedlander. 
In  cases  of  tuberculous  meningitis  the  pus  and  sections  of 
tissue  contain  tubercle-bacilli,  partly  alone  and  partly  in 
association  with  phlogogenic  cocci.  These  bacteria  gain 
access  to  the  cerebral  or  spinal  membranes  from  the  naso- 
pharyngeal space  (lamina  cribrosa),  from  the  middle  ear, 
or  from  the  original  focus  of  suppuration  :  in  the  last  in- 
stance through   the    intermediation    of  the    blood-stream. 


OCCURRENCE  OF  CAUSATIVE   AGENTS.  137 

The  portal  of  entry  for  the  bacteria  in  cases  of  epidemic 
meningitis  has  not  yet  been  determined  with  certainty. 
The  principal  causative  agent  of  this  disease  was,  until 
recently,  believed  to  be  the  diplococcus  lanceolatus  Fran- 
kel,  which  was  found  in  numerous  cases  in  pure  culture. 
A  special  organism  is,  however,  to  be  taken  into  considera- 
tion in  connection  with  the  etiology  of  epidemic  cerebro- 
spinal meningitis — namely,  the  diplococcus  intracellularis 
meningitidis  (Weichselbaum,  Jager).  This  is  a  biscuit- 
shaped  organism,  arranged  in  pairs,  which  is  almost  always 
contained  within  the  cells,  and  which  is  strongly  suggestive 
of  the  gonococcus.  It  is  readily  stained  in  the  exudate, 
but  with  more  difficulty  in  sections.  The  most  suitable 
stain  is  Lofifler's  methylene-blue.  The  organism  does  not 
stain  by  Gram's  method,  although  Jager  maintains  that  it 
does.  It  grows  best  at  37°  C.  (98.6°  F.).  On  agar- 
plates  there  develop  superficial  gray  colonies  that  with 
low  powers  of  the  microscope  appear  dirty-yellow  at  the 
center  and  become  lighter  toward  the  periphery.  The 
deep  colonies  are  quite  small,  and  marked  by  fine  granula- 
tion and  a  slightly  indented  border;  Upon  glycerin-agar 
there  develop  small  gray  colonies  that  at  times  coalesce 
to  form  a  thin  coating.  Upon  blood -serum  a  slight  deposit 
forms.  In  order  to  continue  the  culture  of  meningococci  for 
a  protracted  period  they  must  be  reinoculated  from  every 
four  to  six  days.  They  are  feebly  pathogenic  for  mice  and 
guinea-pigs  on  introduction  into  the  thoracic  or  abdominal 
cavity,  and  for  rabbits  on  introduction  into  the  blood-stream. 

The  clinical  diagnosis  is  based  upon  the  principle  that 
with  the  simultaneous  existence  of  some  other  infectious 
disease — as,  for  instance,  pneumonia,  otitis,  tuberculosis — 
the  causative  agents  of  these  conditions  may  be  considered 
the  cause  of  the  meningitis.  The  absence  of  any  such 
simultaneous  organic  disease  justifies  a  diagnosis  of  epidemic 
cerebrospinal  meningitis. 

A  direct  bacteriologic  diagnosis  is  possible  during  life  by 
means  of  puncture  of  the  spinal  canal,  which  has  now  been 
quite  extensively  practised.  Preparations  are  made  from 
the  exudate,  and  agar-tubes  and  serum-tubes,  or  agar-plates, 
are  inoculated,  and  animals  are  infected.  If  tuberculosis  is 
suspected,  the  specific  stain  for  tubercle -bacilli  must  be  em- 
ployed. To  obtain  the  causative  agents  from  the  meningeal 
pus  after  death,  the  same  plan  is  pursued.     As  the  diplo- 


138  CLINICAL  BACTERIOLOGY. 

COCCUS  is  frequently  encountered,  it  is  advisable  to  inoculate 
a  white  mouse  or  a  rabbit  immediately  with  the  pus. 


BRONCHITIS. 

Bronchitis,  whatever  its  nature  may  be,  is  likewise  de- 
pendent upon  the  activity  of  the  common  exciting  agents  of 
inflammation :  pneumococci,  streptococci,  staphylococci, 
pneumobacilli,  bacterium  coli.  Through  the  action  of 
cold  or  of  some  other  injurious  agency  that  generally  leads 
to  bronchitis,  these  normal  inhabitants  of  the  commence- 
ment of  the  respiratory  tract  become  lodged  in  the  bronchi, 
and  there  excite  inflammation.  Their  demonstration  in 
sputum  is  easy.  The  patient  is  instructed  to  cleanse  his 
mouth  thoroughly  with  a  solution  of  boric  acid  or  of  potas- 
sium chlorate,  and  then  to  expectorate  in  a  sterilized  glass 
dish.  The  sputum  thus  obtained  is  rinsed  carefully  in  sev- 
eral vessels  of  sterile  water,  and  then  a  flake  from  the  center 
of  the  mass  is  smeared  successively  upon  each  of  several 
agar-tubes  or  upon  an  agar-plate  (Koch).  In  this  way  the 
usual  plate -procedure  can  be  avoided,  as  only  one  of  the 
species  of  bacteria  named  is,  as  a  rule,  found  in  the  bron- 
chitic  sputum  in  each  case.  The  large  number  of  bacteria 
that  are  found  on  microscopic  examination  of  the  expectora- 
tion are  mostly  derived  from  the  mouth  and  the  pharynx,  and 
are  adherent,  therefore,  to  the  outer  layers  of  the  sputum. 
In  the  majority  of  cases  there  develop  from  the  expectora- 
tion thus  treated  pure  cultures  of  staphylococci,  Frankel's 
diplococci,  or  streptococci.  If  mixed  infection  is  present, 
the  colonies  develop  separately  in  the  tubes  last  inoculated, 
and  from  these  they  can  be  readily  isolated. 

Fetid  bronchitis  is  associated  with  the  presence  of  the 
same  bacteria,  but,  in  addition,  also  with  putrefactive 
bacteria  (proteus  and  others). 

The  green  color  that  is  sometimes  observed  in  bron- 
chitic  sputum  is  in  some  cases  due  to  the  bacillus  pyocya- 
neus,  in  others  to  the  bacillus  fluorescens,  and  to  varieties 
of  sarcinae. 

PLEURITIS. 

Pleuritis  has  no  uniform  bacteriology.  It  may  be  pri- 
mary or  secondary  ;  in  the  latter  event  in  association  with 
diseases  of  the  lungs,  diseases  of  adjacent  organs,  trauma- 


OCCURRENCE   OF  CAUSATIVE  AGENTS.  139 

tism,  or  general  infection.  The  pleural  effusions  due  to 
general  stasis  (transudates  attending  heart-lesions,  nephritis, 
etc.)  are,  naturally,  sterile,  providing  secondary  infection 
has  not  taken  place,  and  for  which  the  serous  infiltration 
of  the  tissues  offers  a  favorable  soil. 

The  tubercle-bacillus  and  all  of  the  pyogenic  micro- 
organisms are  capable  of  inducing  serous  as  well  as  puru- 
lent pleurisy. 

The  primary  pleurisies  (so-called  pleurisies  due  to  cold) 
are  most  frequently  dependent  upon  the  tubercle-bacillus. 
Next  in  frequency  follows  the  diplococcus  pneumoniae 
Frankel,  which  plays  a  most  important  role,  especially  in 
the  pleural  inflammations  of  childhood  ;  and,  further,  all 
of  the  other  pyogenic  and  phlogogenic  microorganisms. 
Whether  in  all  of  these  cases  the  pleuritic  effusion  is  really 
primary  can  not  always  be  determined  with  certainty,  as 
the  smallest  pulmonary  lesions — for  instance,  slight  disease 
of  the  apex  or  a  bronchopneumonia — that  are  scarcely  sus- 
ceptible of  diagnosis  clinically  may  induce  pleuritis. 

The  secondary  pleurisies  are  associated  in  a  portion  of  the 
cases  with  the  presence  of  the  same  bacteria  that  are 
responsible  for  the  primary  disease.  In  the  effusions  that 
so  frequently  occur  in  the  sequence  of  pneumonia,  in  the 
so-called  metapneumonic  exudates,  there  is  encountered  the 
diplococcus  ;  in  the  effusions  attending  pulmonary  tuber- 
culosis, the  tubercle-bacillus  ;  in  the  uncommon  effusions 
complicating  typhoid  fever,  the  bacillus  of  Gaff  ky-Eberth ; 
in  the  pleurisies  that  originate  in  purulent  processes  within 
the  abdominal  cavity,  the  bacterium  coli ;  and  so  on.  In 
those  diseases  whose  causative  agents  are  as  yet  unknown 
— as,  for  instance,  articular  rheumatism  and  carcinoma — 
the  accompanying  pleural  effusion  has  been  examined  for 
bacteria,  but  as  yet  mostly  with  negative  results.  In  an- 
other portion  of  the  cases  these  concomitant  pleurisies  are 
dependent  upon  secondary  or  upon  mixed  infection.  The 
pleural  effusion  then  contains  the  common  exciting  agents 
of  inflammation.  Thus,  in  the  empyema  following  scarlet 
fever  the  streptococcus  pyogenes  is  often  found  ;  in  that 
following  smallpox,  staphylococci ;  in  that  following  influ- 
enza, the  diplococcus  lanceolatus  Frankel ;  and  so  on. 
Metastatic  pleurisy,  as  part  manifestation  of  a  pyemic 
general  infection,  is  caused  by  the  staphylococcus  or  the 
streptococcus  pyogenes.     The  same  statement  applies  also 


140  CLINICAL  BACTERIOLOGY. 

to  pleurisies  that  arise  in  consequence  of 
wounds  of  the  chest-wall.  The  putrid  effusions  contain, 
in  addition  to  the  causative  agents  of  suppuration,  also 
putrefactive  bacteria,  and  generally  the  proteus. 

Method  of  Bacteriologic  Investigation. — A  hypoder- 
mic syringe  with  a  capacity  of  from  one  to  six  cubic  centi- 
meters is  kept  filled  for  from  six  to  twelve  hours  in  five 
per  cent,  carbolic  acid,  and  is  then  carefully  cleansed  with 
sterilized  water  in  order  to  remove  all  of  the  disinfectant ; 
or  a  Roux's  syringe  is  sterilized  by  thorough  boiling.  The 
point  on  the  chest-wall  where  the  exploratory  puncture  is 
to  be  made  is  washed  with  soap,  alcohol,  mercuric-chlorid 
solution  (i  :  looo),  and  ether,  and  the  puncture,  after 
thorough  disinfection  of  the  hands,  is  made  in  the  usual 
way.  The  fluid  obtained  is  received  into  a  sterilized  dish  and 
each  of  four  or  five  agar-tubes  successively,  or  an  agar-plate, 
is  inoculated  with  a  drop  thereof,  or  a  drop  of  the  exudate 
is  permitted  to  flow  directly  from  the  syringe  upon  the  sur- 
face to  be  inoculated.  The  tubes  are  introduced  into  the 
thermostat  at  a  temperature  of  37°  C.  (98.6°  F.).  At  the 
same  time  cover-slip  preparations  are  made  in  the  usual 
manner,  and  examined  for  tubercle-bacilli  and  other  bac- 
teria. With  the  remainder  of  the  fluid,  if  tuberculosis  be 
suspected,  two  or  three  guinea-pigs  may  be  inoculated 
through  the  peritoneum  (see  later).  Serous  effusions  con- 
tain kw,  if  any,  microorganisms.  It  is,  therefore,  a  more 
reliable  procedure  to  remove  considerable  amounts  of  the 
fluid,  to  centrifugate  or  sediment  it,  and  to  study  the  pre- 
cipitate only. 

Diagnostic  and  Prognostic  Significance  of  the  Bac- 
teriologic Findings. — Bacteriologic  investigation  is  not  of 
great  importance  in  the  diagnosis  of  serous  effusions.  The 
large  majority  of  serofibrinous  pleurisies  prove  to  be  sterile. 
The  metapneumonic  serous  effusions  contain  at  times  the 
diplococcus  lanceolatus  Frankel,  and  before,  as  well  as 
after,  the  crisis.  The  presence  of  pyogenic  microbes  in 
serous  pleural  effusions,  as  has  been  repeatedly  observed, 
does  not,  in  all  instances,  justify  the  conclusion  that  puru- 
lent metamorphosis  into  an  empyema  will  take  place.  Such 
effusions  may,  under  circumstances,  recede  completely.  If 
a  decision  is  to  be  reached  in  doubtful  cases  of  serofibrinous 
pleurisy  whether  tuberculosis  exists  or  not,  it  is  advisable 
to  inject  into  the  peritoneum  of  guinea-pigs  some  of  the 


OCCURRENCE  OF  CAUSATIVE   AGENTS.  141 

pleural  fluid  obtained  under  sterile  conditions,  and  to  wait 
and  see  whether  the  animals  die  of  tuberculosis  or  not. 
This  procedure,  however,  always  occupies  several  weeks, 
and  even  then  it  is  not  always  entirely  reliable,  and  it  fre- 
quently fails  to  resolve  the  doubt,  as  in  spite  of  an  evident 
tuberculous  origin  of  the  pleuritic  effusion  the  animals  often 
remain  well.  The  reason  for  this  is  to  be  found  in  the 
small  number  in  which  the  tubercle-bacilli  are  usually 
present  in  the  effusion.  It  is,  therefore,  advisable  to  centrif- 
ugate  a  considerable  amount  of  the  effusion,  and  to  inocu- 
late guinea-pigs  with  the  precipitate  thus  obtained. 

Bacteriologic  examination  is,  however,  much  more  im- 
portant in  the  diagnosis  of  empyema.  If  the  agar-tubes 
inoculated  with  the  pleural  pus  remain  sterile,  this  indicates 
that  the  process  is,  in  all  probability,  tuberculous.  Direct 
microscopic  demonstration  of  tubercle-bacilli  in  cover-glass 
preparations  is,  however,  successful  only  in  a  few  cases. 
Not  rarely  the  empyema  of  tuberculous  subjects  is  due  to 
secondary  infection. 

Various  observers  have  reached  conclusions  with  regard 
to  both  the  prognosis  and  the  treatment  of  empyema  from 
the  species  and  the  virulence  of  the  bacteria  found  in  the 
pleural  pus.  The  presence  of  the  diplococci  is  believed  to 
indicate  a  much  more  favorable  prognosis  than  that  of  the 
other  pyogenic  organisms.  In  this  so-called  pneumococcus- 
empyema  less  radical  treatment  is  therefore  necessary. 
Thoracotomy  would,  under  these  circumstances,  be  super- 
fluous, simple  evacuation  by  puncture  being  sufficient. 
It  can  actually  be  admitted  that  the  prognosis  of  pneumo- 
coccus-empyema  is  usually  good.  Nevertheless,  radical 
operation  is  indicated  if  the  effusion  is  not  quickly  absorbed 
spontaneously,  or  at  least  after  puncture.  Too  much  reli- 
ance is  not  to  be  placed  upon  spontaneous  attenuation  of 
the  bacteria  in  the  pus,  or  upon  their  death,  although  the 
organisms  in  question  live  usually  only  for  a  short  time  in 
artificial  culture.  Bacteria  with  fully  preserved  virulence  have 
been  cultivated  from  pleural  pus  after  the  lapse  of  as  long 
as  three  and  one-half  months.  Further,  recovery^  from  em- 
pyema due  to  staphylococci,  streptococci,  and  even  typhoid 
bacilli,  has  been  reported  in  isolated  instances  as  occurring 
spontaneously  or  after  simple  puncture.  The  question  is 
yet  undecided  whether  operation  should  be  undertaken  for 
the  relief  of  purulent  effusions  in  tuberculous  subjects  when 


142  CLINICAL  BACTERIOLOGY. 

they  contain  tubercle-bacilli  or  are  sterile.  While  operation 
for  all  other  varieties  of  empyema,  even  in  cases  of  tuber- 
culosis, is  attended  with  a  relatively  favorable  prognosis, 
this  is  unfavorable  in  cases  of  empyema  associated  with  the 
presence  of  tubercle-bacilli.  Generally,  healing  does  not  take 
place,  a  fistula  remains,  and  the  chronic  suppuration  leads 
to  death.  Nevertheless,  some  surgeons  recommend  opera- 
tion even  in  these  cases,  as,  without  doubt,  recovery  may 
take  place  also  under  such  conditions. 

Pneumothorax. — Pneumothorax  with  perforation,  which 
occurs  so  commonly  in  tuberculosis,  is,  as  a  rule,  followed 
by  purulent  effusion.  The  exudate  contains  tubercle- 
bacilli,  demonstrable  microscopically  or  by  experiment  on 
animals,  and,  in  consequence  of  mixed  infection,  one  or 
another  of  the  pyogenic  cocci,  and,  besides,  at  times,  putre- 
factive bacteria,  especially  the  proteus.  Only  one  case  of 
pneumothorax  without  perforation  has  been  studied  bacter- 
iologically.  In  this  the  anaerobic,  gas-forming  bacillus 
emphysematosus  (p.  128)  was  found  to  be  the  exciting 
agent.  The  method  of  examination  is  the  same  as  in  the 
case  of  pleurisy. 

PNEUMONIA. 

Frankel's  pneumococcus  maybe  looked  upon  as  the  caus- 
ative agent  of  lobar  croupous  pneumonia,  as  this  organism 
is  demonstrable  in  more  than  three-fourths  of  all  the  cases 
in  the  pulmonary  tissues,  which,  normally,  are  entirely  free 
from  bacteria.  In  the  larger  proportion  of  cases  the  pneu- 
mococcus alone  is  present  in  the  diseased  tissue  ;  in  the 
smaller  proportion,  staphylococci  and  streptococci  besides 
are  present.  Streptococci  alone,  or  in  association  with 
staphylococci,  rarely  staphylococci  alone,  may,  however, 
be  found  in  pneumonic  foci.  In  addition,  the  bacillus  of 
Friedlander  (Figs.  42,  47)  is  found  in  some  cases.  All  of 
these  bacteria,  as  has  been  pointed  out,  exist  in  the  upper  air- 
passages.  They  have,  further,  been  demonstrated,  though 
less  commonly,  in  the  healthy  larynx,  but  bacteria  do 
not  occur  in  the  bronchi  and  in  the  pulmonary  tissues  in 
healthy  persons.  For  the  development  of  pneumonia,  it  is, 
therefore,  generally  necessary  that  an  accidental,  indirect 
cause  (cold,  traumatism,  etc.)  shall  cooperate  that  renders 
it  possible  for  the  bacterium  to  penetrate  more  deeply 
into  the  air-passages,  and  there  to  give  rise  to  inflammation. 


.*^^ 


>^ 


OCCURRENCE  OF  CAUSATIV] 


IZtspartjrf^ 


It  is  possible,  to  a  certain  degree,  to  differentiate  the 
pneumonias  according  to  their  causative  agents  : 

I.  Pneuniococcus-pneumonia  corresponds  with  the  charac- 
teristic cHnical  picture  of  true  croupous  pneumonia,  with 
fibrinous  exudation  into  the  alveoli,  and  lobar  distribution 
of  the  morbid  process.  Clinically,  this  variety  is  character- 
ized by  blood-streaked  sputum,  and  especially  by  the 
sudden  onset  of  the  disease  with  a  chill,  the  high,  stormy 
course,  and  the  critical  decline  of  the  fever.  That  this 
special  type  of  disease  is  related  etiologically  to  the  activity 
of  pneumococci   is   proved  almost  with    certainty  by  the 


Fig.  47. — Bacillus  pneumoniae  of  Friedlander,  from  the  expectoration  in  a  case  of 
pneumonia  ;  X  1000  (Frankel  and  Pfeiffer). 


following  facts  :  In  the  first  place,  in  typical  cases  the 
diplococcus  alone  is  always  found  in  the  diseased  area,  in 
which  it  is  demonstrable  during  life  by  puncture.  In  the 
next  place,  the  general  symptoms  characteristic  of  pneu- 
monia are  also  often  present  in  marked  degree,  in  con- 
junction with  other  localizations  of  the  diplococcus,  espe- 
cially pneumococcus-angina.  Finally,  it  may  be  pointed  out 
in  this  place  that  the  blood  of  human  beings  that  have 
passed  through  an  attack  of  typical  pneumonia  with 
critical  defervescence  is  capable  of  immunizing  animals  to 
pneumococci.  There  is  thus  furnished  evidence  that  con- 
valescents from  pneumonia  are  often  immune   to  pneumo- 


144  CLINICAL   BACTERIOLOGY. 

cocci,  and  it  is  thus  comprehensible  why  the  symptoms  dis- 
appear abruptly  with  the  occurrence  of  the  crisis,  while  the 
pneumonic  infiltration  remains '  unchanged.  From  these 
facts  it  may  be  inferred  that  a  toxic  action  on  the  part  of  the 
pneumococci  constituted  a  decisive  element  in  the  previous 
clinical  picture ;  for,  when  the  crisis  at  once  obliterates  the 
disease,  no  change  has  been  effected  in  its  anatomic  basis, 
in  the  infiltrate  itself;  only  the  toxic  activity  of  the  pneu- 
mococci has  been  withdrawn,  and  the  manifestations  that 
have  just  disappeared  may,  therefore,  reasonably  be  referred 
to  that. 

Finally,  it  may  be  mentioned  that  true  pneumonia  has 
been  induced  experimentally  in  favorable  cases  by  inhalation 
of  pneumococci.  Such  experiments  are  usually  attended 
with  difficulty,  because  the  animals  commonly  employed 
(rabbits,  mice),  which  are  so  much  more  susceptible  to  the 
pneumococcus  than  human  beings,  die,  as  a  rule,  in  conse- 
quence of  the  septicemic  general  infection,  and  localized 
disease  is  induced  in  them  with  difficulty. 

2.  Streptococcus-pneumonia  corresponds  with  the  clinical 
picture  of  bronchopneumonia  (cellular,  catarrhal  pneu- 
monia). The  infiltrate  is  only  lobular,  usually  less  dense, 
and  associated  with  less  fibrin  ;  the  sputum  is  mucopuru- 
lent and  not  rusty ;  the  onset  is  not  so  pronounced  ;  the 
course  is  more  insidious  and  attended  with  remissions  and 
intermissions  in  the  fever  (so-called  streptococcus-curve)  ; 
and,  above  all,  the  crisis  is  wanting.  Some  cases  of  strepto- 
coccus-pneumonia are  characterized  by  especial  severity 
iypneumonie  infectieuse  of  the  French).  The  differentiation 
of  this  variety  is,  however,  not  readily  sustained,  in  the 
first  place  because — 

3.  Staphylococcus-pneumonia,  which  is  exceedingly  un- 
common as  a  single  infection,  resembles  it  absolutely ;  and, 
secondly,  because  of  the  occurrence. so  frequently  of — 

4.  Mixed  forms  of  pneumojiia,  in  which  two  or  even  three 
of  the  bacteria  named  are  found  together.  These  forms  of 
the  disease  furnish  clinically  also  a  mixed  picture  that 
occupies  a  position  between  croupous  pneumonia  and 
bronchopneumonia.  As  a  rule,  the  occurrence  of  blood- 
streaked  sputum  or  the  temperature-curve,  even  when  the 
infiltrate  is  small,  indicates  the  participation  of  pneumo- 
cocci in  the  morbid  process  ;  or,  in  a  case  of  apparently 
true  croupous   pneumonia,  which,  however,  is   not  a  pure 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  145 

type  etiologically,  but  is  a  mixed  form,  the  crisis  does  not 
appear.  On  the  whole,  however,  there  are  no  fixed  rules 
for  these  mixed  types.  They  render  impossible  the  clinical 
diagnosis  with  regard  to  the  causative  agent  in  the  indi- 
vidual case. 

5.  Influenza-pnemnonia  is  associated  with  the  presence 
of  the  influenza-bacillus,  partly  in  pure  culture,  and  partly 
together  with  streptococci  or  pneumococci. 

6.  The  pneumonia  complicating  tuberculosis  in  the  form 
of  caseous  pneumonia  may  be  due  to  the  tubercle-bacillus 
exclusively.  On  the  other  hand,  pneumonia  dependent 
upon  streptococci,  pneumococci,  and  influenza-bacilli  is, 
however,  not  at  all  uncommon  in  tuberculous  lungs. 
When  pneumococci  take  part  in  the  morbid  process,  the 
clinical  picture  assumes  the  peculiar  characters  that  have 
been  described,  and  are  indicative  of  an  acute  severe  infec- 
tion. 

7.  The  pneumonia  complicatiiig  other  infectious  diseases  is 
less  commonly  due  to  the  cause  of  those  diseases — as,  for 
instance,  typhoid  pneumonia  due  to  typhoid-bacilli — and 
more  commonly  to  the  organisms  usually  causative  of  in- 
flammation. The  pneumonia  that  occurs  in  the  sequence 
of  diseases  of  the  abdominal  organs  is  often  associated  with 
the  presence  of  the  bacterium  coli — as,  for  instance,  the 
bronchopneumonia  not  rarely  observed  in  connection  with 
incarcerated  hernia. 

The  bacteriologic  diagnosis  can  be  made  from  exam- 
ination of  the  sputum,  but  the  procedure  of  Koch,  as  de- 
scribed by  Kitasato,  for  the  culture  of  tubercle-bacilli 
directly  from  the  sputum  must  be  observed — that  is,  the 
mass  of  sputum  must  be  thoroughly  rinsed  in  dishes  with 
sterilized  water,  in  order  to  remove  the  large  number  of 
bacteria  that  have  been  added  from  the  pharynx  and  the 
mouth.  Then  a  flake  is  isolated  from  the  interior  of  the 
mass,  and  this  is  smeared  several  times  upon  an  agar-plate 
or  upon  several  glycerin-agar  tubes  in  slants  ;  that  which  is 
left  is  examined  microscopically. 

Frequently  direct  puncture  of  the  diseased  lung  has  been 
made  with  a  thoroughly  sterilized  hypodermic  syringe, 
and  a  few  drops  of  the  exudate  are  obtained  ;  and  these  are 
treated  bacteriologically  in  the  manner  described.  This 
procedure — perfect  asepsis  being  observed — need  not  occa- 
sion the  slightest  concern,  as  it  is  often  practised  involun- 


146  CLINICAL  BACTERIOLOGY. 

tarily  in  the  search  for  pleuritic  effusions  (exploratory 
puncture)  without  ever  doing  any  harm. 

If  the  information  be  sought  whether  the  pneumococcus 
participates  in  the  process,  it  is  best  to  inject  subcuta- 
neously  some  of  the  sputum  or  pulmonary  fluid  into  a  mouse, 
or,  better,  into  the  still  more  susceptible  rabbit.  If  the 
pneumococcus  is  present,  the  animal  will  die  in  from  twenty- 
four  to  forty-eight  hours  in  consequence  of  diplococcus- 
septicemia,  which  is  readily  demonstrable  microscopically 
in  stained  cover-glass  preparations  from  the  heart's  blood 
of  the  animal. 

Prognostic  significance  is  to  be  attached  in  only  limited 
degree  to  the  results  of  bacteriologic  examination,  be- 
cause in  the  case  of  pneumonia  also  much  depends  upon 
the  virulence  of  the  exciting  agent.  However,  a  crisis  may 
be  looked  for  with  greater  assurance  when  pneumococci 
alone  are  found  than  under  any  other  conditions.  It  must, 
however,  not  be  forgotten  that  even  in  cases  of  pure  pneu- 
mococcus-pneumonia  the  crisis  may  not  occur ;  while,  on 
the  other  hand,  it  may  be  present  in  cases  of  streptococcus- 
pneumonia.  The  general  condition  of  the  patient,  evidently, 
is  of  considerable  importance  in  this  connection,  and  it 
must  be  given  due  consideration  in  every  aspect  in  pre- 
dicting the  crisis.  Thus,  the  prognosis  is  unfavorable  in 
the  pneumonia  of  alcoholics,  in  that  of  the  aged,  and  of 
those  with  kyphosis,  even  when  pneumococci  alone  are 
found.  The  patients  often  die  before  the  crisis,  or  the  dis- 
ease pursues  a  protracted  course,  and  terminates  late  with- 
out the  occurrence  of  a  crisis.  The  pneumococcus-pneu- 
monia  of  tuberculous  subjects  justifies  a  more  favorable 
prognosis  than  a  purely  tuberculous  pneumonia  under  the 
same  conditions. 

Reference  has  already  been  made  to  the  conception  of 
the  crisis  as  indicating  the  advent  of  immunity,  as  well  as 
to  the  evidence  of  the  immunizing  property  of  the  blood- 
serum  after  the  crisis  in  a  number  of  cases  of  pneumonia. 
Why  this  immunity  is  so  transient,  disappearing  in  some 
cases  in  the  course  of  a  few  days,  is  not  yet  known.  In 
any  event  it  has  been  established  clinically  that  pneumonia 
has  a  tendency  to  recur,  and  it  may  almost  be  included 
among  those  diseases  of  which  one  attack  rather  predisposes 
to  subsequent  attack.  In  a  pneumococcus-culture  the 
bacteria  die  in  the  course  of  a  few  days  (from  four  to  seven). 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  147 

but  this  takes  place  only  in  cultures,  and  not  in  the  body, 
and  it  is  incapable,  therefore,  of  explaining  the  crisis.  The 
focus  of  disease  in  the  lung,  as  well  as  the  sputum,  contains 
living  bacteria  both  during  and  long  after  the  occurrence  of 
the  crisis.  Often  these  retain  their  virulence  throughout  the 
entire  period.  Should  the  virulence  be  diminished  during 
the  crisis,  it  is,  however,  soon  again  augmented,  as  experience 
has  shown.  It  is,  therefore,  not  the  cocci,  but  the  human 
organism  that  undergoes  some  change  in  the  crisis  :  it  be- 
comes insusceptible  to  the  diplococcus-virus — that  is,  im- 
mune. 

Transmission  of  Pneumonia. — Infection  with  pneumonia 
takes  place  principally  through  the  respiratory  passages. 
Only  in  some  cases  of  secondary,  complicating  pneumonia 
may  the  microorganisms  gain  entrance  into  the  lungs 
through  the  blood-stream.  Direct  transmission  of  pneu- 
monia from  one  individual  to  another  appears  possible,  and 
a  considerable  number  of  Jiouse-cpide7nics  of  pneumonia 
have  been  reported.  In  the  majority  of  cases  an  attack  of 
pneumonia  probably  results  from  the  inhalation  of  pneumo- 
cocci  with  the  air ;  or,  more  frequently,  pneumococci  that 
have  long  been  present  in  the  mouth,  the  pharynx,  or  the 
nose,  are  permitted  by  some  accidental  occurrence  to  gain 
entrance  into  the  lungs  and  there  to  set  up  inflammation. 
Naturally,  in  connection  with  the  epidemic  distribution  of 
pneumonia,  the  possibility  can  not  be  excluded  that  the 
accidental  influence  has  been  operative  in  all  cases  in  com- 
mon, that  the  pneumococcus  need  not  be  transmitted  from 
one  person  to  another,  but  that  it  was  present  previously  in 
all  affected. 

The  hereditary  transmission  of  pneumonia  from  the 
mother  to  the  fetus  is  a  matter  of  great  interest.  A  few 
cases  have  been  reported  in  which  the  children  of  pneu- 
monic mothers  have  been  born  with  pneumonia.  In  gen- 
eral the  pneumococcus  is  not  disseminated  outside  the 
lungs  of  the  infected  individual.  The  slighter  degree  of 
susceptibility  of  human  beings  protects  them,  as  a  rule, 
against  septicemic  infection,  which  occurs  regularly,  for  in- 
stance, in  the  more  susceptible  rabbit.  In  some  cases, 
however,  the  pneumococcus  has  been  found,  also,  in  the 
blood  of  pneumonic  patients  during  life.  Such  cases  have 
always  been  marked  by  especial  severity,  with  a  fatal  ter- 
mination, so  that  the  discovery  of  pneumococci  in  the  cir- 


148  CLINICAL  BACTERIOLOGY. 

culating  blood  must  always  be  considered  as  of  unfavorable 
prognostic  omen.  In  the  small  number  of  cases  of  con- 
genital pneumonia  reported  the  bacteria  must  have  gained 
entrance  into  the  placenta,  through  a  lesion  of  which  they 
have  passed  over  into  the  fetal  circulation.  Without 
such  lesion  the  placenta,  as  is  quite  generally  admitted,  is 
impassable  to  microorganisms.  Like  all  febrile  diseases, 
pneumonia  readily  induces  abortion.  The  child  that  has 
been  infected  with  diplococci  can,  however,  suffer  from 
true  pneumonia  only  if  it  has  already  breathed.  As  a 
matter  of  fact,  in  only  two  of  the  children  in  the  reported 
cases  was  pneumonic  infiltration  present ;  in  the  others 
diplococci  were  found  in  the  blood — there  existed  a  sep- 
ticemia. 

ENDOCARDITIS. 

Endocarditis  occurs  usually  as  a  secondary  infection  in 
the  course  of  various  other  diseases.  The  most  important 
of  these  is  acute  articular  rheumatism,  whose  predominant 
etiologic  significance  in  the  generation  of  endocarditis  is 
well  known.  The  causative  agents  of  rheumatism  have 
not  yet  been  isolated,  and  rheumatic  endocarditis  also  is 
one  of  those  infectious  diseases  whose  intimate  etiology  re- 
mains to  be  cleared  up.  The  same  statement  may  be  made 
with  regard  to  chorea,  whose  endocarditic  manifestations 
also  are  probably  to  be  attributed  to  the  same  rheumatic 
etiology ;  and  also  with  regard  to  erythema  nodosum. 

In  the  deposits  upon  the  valvc3  of  the  heart  in  the  se- 
quence of  erysipelas  the  streptococcus  pyogenes  is  demon- 
strable both  on  culture  and  on  microscopic  examination. 

In  the  endocarditis  following  suppurative  processes  (py- 
emia, septicemia,  puerperal  fever)  the  streptococcus  or  the 
staphylococcus  pyogenes  has  been  found  in  the  vegetations  : 
in  that  following  osteomyelitis,  generally  the  staphylo- 
coccus ;  and  after  suppuration  in  the  abdominal  cavity,  the 
bacterium  coli  commune. 

Endocarditis  is  comparatively  frequent  in  the  sequence  of 
croupous  pneumonia.  The  lesions,  which  preferably  involve 
the  aortic  valve,  contain,  as  a  rule,  the  diplococcus  lanceo- 
latus  Frankel.  Bacteriologic  examinations  with  reference 
to  the  endocarditis  following  influe7iza  are  yet  wanting. 

Diphtheric  endocarditis  is  extremely  uncommon.  Only 
one  case  of  true  diphtheric  endocarditis  (mitral  valve),  with 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  149 

demonstration  of  diphtheria-bacilli,  has  been  recorded  in 
the  literature. 

Almost  equally  as  uncommon  as  diphtheric  endocarditis 
is  also  true  typhoid  endocarditis — that  is,  that  caused  by  the 
Gaffky-Eberth  bacillus. 

Tubercidous  endocarditis  has  long  been  known.  It  attacks 
with  a  certain  degree  of  exclusiveness  the  thin  margins  of 
the  mitral  leaflets,  and  particularly  on  their  auricular  sur- 
face. Tubercle-bacilli  have  been  repeatedly  demonstrated 
in  the  lesions. 

True  gonorrheal  endocarditis  is  dependent  upon  the  activ- 
ity of  the  gonococcus,  whose  presence  in  the  endocarditic 
vegetations  has  been  demonstrated  by  Ley  den. 

The  causative  agent  of  the  endocarditis  following  measles, 
scarlet  fever,  smallpox,  as  well  as  the  causes  of  the  primary 
disease,  are  still  unknown. 

The  endocarditis  complicating  acute  nephritis  is  generally 
caused  by  the  same  microorganisms  (the  exciting  agents  of 
suppuration  and  inflammation)  that  give  rise  to  the  primary 
morbid  process  in  the  kidneys. 

It  is,  however,  to  be  emphasized  that  in  all  of  the  dis- 
eases named,  as  well  as  in  malaria  and  in  carcinoma,  the 
concomitant  endocarditis  need  not  be  due  to  the  original 
infecting  pathogenic  germ,  but  it  may  be  the  expression  of 
a  secondary  or  of  a  mixed  infection  that  has  been  engrafted 
upon  the  endocardium,  whose  resistance  has  been  lowered 
by  reason  of  the  primary  infection.  Under  such  circum- 
stances staphylococci,  streptococci,  diplococci,  or  the  bac- 
terium coli  commune  will  be  found  in  the  vegetations  upon 
the  valves  :  in  short,  those  microorganisms  that  have  re- 
peatedly been  shown  to  be  the  cause  of  secondary  infection. 

So-called  malignant,  ulcercLtive  endocarditis  is  etiologi- 
cally  and  clinically  only  a  variety  of  ordinary  endocarditis 
pursuing  a  malignant  course,  and  giving  rise  to  necrosis  of 
the  vegetations.  If  it  is  due  to  the  common  exciting  agents 
of  inflammation — staphylococci,  streptococci,  pneumococci, 
or  the  bacterium  coli  commune — it  may  be  viewed  as  a 
special  form  of  pyemia,  characterized  by  the  localization 
of  the  metastases  on  the  valves  of  the  heart.  In  isolated 
cases  of  this  kind  the  microorganisms  were  demonstrated 
in  the  circulating  blood  during  life. 

Experimental  Development  of  Endocarditis  by  Means 
of  Bacteria. — After  a  previous  lesion  of  the  valves  (through 


,  150  CLINICAL  BACTERIOLOGY. 

catheterization  by  way  of  the  right  carotid),  it  has  been 
possible  to  induce  endocarditic  changes  of  maHgnant  char- 
acter in  experiments  on  animals  by  intravenous  injection 
of  the  exciting  agents  of  inflammation  and  suppuration. 
The  same  result  may  be  attained  without  this  serious  pro- 
cedure if  the  bacterial  material  is  introduced  into  the  vein 
in  the  form  of  a  suspension  that  contains  coarse  particles — 
for  instance,  from  a  potato-culture.  The  particles,  with  the 
contained  and  adherent  microorganisms,  lodge  upon  the 
valves,  and  thus  form  the  starting-point  of  endocarditic 
changes. 

Bacteriologic  Diagnosis. — In  doubtful  cases  it  is  advis- 
able to  examine  the  blood  bacteriologically  (p.  162).  As 
has  been  mentioned,  it  is  sometimes  possible  to  demonstrate 
the  microorganisms  in  the  circulating  blood,  and  the  diag- 
nosis of  ulcerative  endocarditis  is  then  justified.  In  the 
majority  of  cases,  however,  examination  of  the  blood  yields 
negative  results,  and  even  in  such  cases  as  exhibit  a  dis- 
tinctly malignant  character.  The  clinical  diagnosis  may, 
therefore,  receive  but  little  support  from  bacteriologic  ex- 
amination in  connection  with  this  disease. 


PERICARDITIS. 

Also  with  regard  to  pericarditis,  the  distinction  between 
primary  and  secondary  inflammation  can  not  readily  be 
made  from  the  etiologic  standpoint.  The  principal  factor 
in  the  causation  of  pericarditis  likewise  is  acute  articular 
rhezunatism,  whose  exciting  agent,  as  has  been  stated,  is 
unknown.  Traumatic  pericarditis  is  either  to  be  referred 
to  the  action  of  bacteria  that  gain  entrance  into  the  peri- 
cardial cavity  simultaneously  with  the  inception  of  the 
injury,  or,  in  the  case  of  nonpenetrating  wounds  of  the 
thorax,  a  point  of  lessened  resistance  is  established  in  the 
pericardium  in  consequence  of  rupture  of  blood-vessels, 
ecchymosis,  etc.,  resulting  from  the  contusion  of  the  chest, 
in  which  microorganisms  circulating  in  the  blood  lodge 
and  give  rise  to  inflammation.  Pericarditis  in  the  sequence 
of  erysipelas  is  dependent  upon  the  streptococcus  pyogenes  ; 
the  pericarditis  attending  pnetimonia,  upon  the  diplococcus 
of  Frankel  ;  puerperal  and  pyemic  pericarditis,  upon  the  ex- 
citing agents  of  suppuration. 

A  distinct  position  is  occupied  by  tuberculous  pericarditis ^ 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  151 

which,  next  to  the  rheumatic,  is  probably  the  most  com- 
mon variety.  Its  exciting  agent,  the  tubercle-bacillus, 
gains  entrance  into  the  pericardium  through  direct  exten- 
sion of  the  tuberculous  process  from  the  adjacent  lung,  or 
by  way  of  the  blood  or  the  lymph. 

The  bacteriologic  diagnosis  is  possible  during  life  only 
on  puncture  or  operation  for  the  relief  of  the  pericardial 
effusion. 

MYOCARDITIS. 

Suppurative  Myocarditis. — A  greater  or  smaller  number 
of  purulent  foci  of  varying  size  may  be  present  in  the  heart- 
muscle  in  association  with  pyemic  processes  of  diverse 
origin.     They  contain  pyogenic  microbes. 

Acute  Diffuse  Myocarditis. — Acute  inflammation  of  the 
myocardium  may  complicate  all  infectious  diseases  of  rapid 
course.  Its  bacteriologic  relations  are  as  yet  but  little 
known.  The  bacillus  of  Gaffky-Eberth  has  been  repeat- 
edly found  in  the  myocardium  in  cases  of  typhoid  fever. 
Frequently,  however,  the  infection  appears  to  be  secondary 
and  dependent  upon  the  common  exciting  agents  of  inflam- 
mation. The  metabolic  products  of  microorganisms  may 
likewise  ^w^  rise  to  similar  myocarditic  alterations — as,  for 
instance,  the  toxin  of  the  diphtheria-bacillus,  which  is  the 
cause  of  diphtheric  myocarditis. 


PERITONITIS. 

The  inflammatory  processes  involving  the  peritoneum 
may  be  induced  through  chemic  influences  exerted  by  the 
metabolic  products  of  bacteria — aseptic  peritonitis,  for  in- 
stance, in  consequence  of  absorption  of  decomposition- 
products  from  the  intestine  in  cases  of  ileus.  In  the 
majority  of  cases,  however,  such  processes  result  directly 
from  the  activity  of  bacteria  {bacterial  or  septic  peritonitis^. 
In  accordance  with  the  source  of  the  exciting  agents,  peri- 
tonitis is  conveniently  divided  into  several  varieties  :  (i) 
That  which  arises  from  the  digestive  tract  —  from  the 
stomach,  the  duodenum,  and  the  small  intestine,  as  well  as 
from  the  cecum,  the  vermiform  appendix,  the  colon,  and  the 
rectum  ;  (2)  that  which  arises  from  the  gall-bladder  and 
the  liver ;  (3)  that  which  arises  from  the  kidneys  and  the 
urinary  bladder ;    (4)  that   which   arises    from   the  female 


152  CLINICAL   BACTERIOLOGY. 

genito-urinary  tract ;  to  these  may  be  added  (5)  the  rare 
cases  in  which  the  infection  is  of  hematogenous  origin  ;  and 
(6)  the  still  rarer,  in  which  the  peritonitis  is  the  result  of 
operative  intervention.  In  the  first  four  groups  named  the 
bacteria  may  migrate  to  the  peritoneum  (as  in  the  case  of 
puerperal  peritonitis,  in  which  the  exciting  agents  reach  the 
peritoneum  from  the  uterus  by  way  of  the  lymphatics)  in 
the  absence  of  a  breach  in  the  continuity  of  the  organ 
primarily  infected  ;  or  there  may  be  such  a  breach  in  con- 
tinuity, and  perforative  peritonitis  results.  The  latter  is  the 
more  dangerous  variety,  because  with  the  microorganisms 
other  materials — as,  for  instance,  intestinal  contents — gain 
entrance  into  the  peritoneal  cavity  through  the  perfora- 
tion, and  these  act  as  chemic  and  mechanical  irritants, 
and  thus  make  possible  and  easy  the  proliferation  of  the 
bacteria.  By  reason  of  the  extraordinarily  great  absorp- 
tive power  and  the  extent  of  the  absorbing  surface  of  the 
peritoneum,  the  entrance  of  bacteria  into  the  peritoneal 
cavity  in  experiments  on  animals  does  not  invariably  lead 
to  peritonitis,  as  has  been  demonstrated  by  the  injection  of 
moderate  amounts  of  pyogenic  cocci  into  the  abdominal 
cavity  of  -dogs,  rabbits,  and  guinea-pigs.  For  the  develop- 
ment of  peritonitis  a  predisposing  factor  is  additionally 
necessary,  such  as  especial  virulence  of  the  bacteria — -as, 
for  instance,  in  general  septicemia — the  simultaneous  en- 
trance of  intestinal  contents,  and,  experimentally,  the  con- 
joint introduction  of  considerable  amounts  of  preformed 
bacterial  poisons,  etc. 

The  distinction  between  diffuse  and  circumscribed  peri- 
tonitis, which  is  so  important  clinically,  is  not  supported  by 
bacteriologic  examination,  as  the  causative  agents  are  the 
same  in  both  instances. 

The  bacteriologic  diagnosis  can  only  be  made  in  con- 
nection with  operation  or  on  exploratory  puncture.  If  it 
is  desired  for  special  reasons  in  a  given  case  to  make  an 
examination  for  bacteria  after  death,  this  must  be  done  as 
soon  as  possible  after  dissolution  has  taken  place,  as  the 
result  may  be  invalidated  by  the  migration  of  bacteria  from 
the  intestine  after  the  lapse  of  a  few  hours.  From  the 
effusion  or  from  the  deposits  upon  the  peritoneum — in  ad- 
dition to  microscopic  examination — plates  are  simply  cast 
or  tube-slants  are  smeared. 

The  many  bacteriologic  investigations   that  have   been 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  153 

carried  out  in  the  study  of  peritonitis  have  thus  far  not 
yielded  practically  available  results.  In  some  of  the  cases 
there  existed  multiple  infection — that  is,  infection  with  a 
number  of  varieties  of  bacteria.  Coli-bacteria,  as  well  as 
staphylococci  and  streptococci,  have  been  mostly  found  in 
the  effusion.  In  addition,  pneumococci  have  been  ob- 
served, as  well  as  a  large  number  of  other  bacteria,  of  which 
the  gonococcus,  the  proteus  vulgaris,  the. bacillus  pyocya- 
neus,  as  well  as  a  number  of  nonpathogenic  varieties 
described  by  Tavel  and  Lanz,  a  bacillus  resembling  that  of 
diphtheria,  one  resembling  the  tetanus-bacillus,  and  one  the 
actinomyces-fungus,  may  be  mentioned.  The  bacterium 
coli  commune,  which  is  the  most  common  attendant  upon 
peritonitis,  is  not  a  bacteriologic  entity,  but  so  variable  in 
form  and  properties  that  the  designation,  bacterium  coli, 
must  be  employed  only  as  the  collective  name  for  a  large 
group  of  related  bacteria.  When  infection  takes  place 
through  the  blood-stream,  only  one  exciting  agent  is 
found,  and  principally  the  streptococcus  or  the  pneumo- 
coccus.  In  cases  of  operative  peritonitis,  usually  strepto- 
cocci have  been  found  ;  and  in  cases  of  puerperal  peritonitis, 
in  addition  also  the  other  exciting  agents  of  inflammation. 
Tuberculous  peritonitis  is  caused  by  the  tubercle-bacillus. 

From  all  that  has  been  said  it  may  be  seen  that  peri- 
'tonitis  is  not  due  to  a  single  specific  exciting  agent,  as  the 
organ  from  which  it  arises  (bladder,  intestine)  generally 
contains  various  bacteria.  The  infections  that  originate  from 
the  female  genital  organs  are  associated  especially  with 
cocci ;  those  originating  from  the  intestine,  especially  with 
coli-bacteria.  Further,  the  peritonitis  arising  from  the 
small  intestine  is  believed  to  be  attended  with  the  presence 
of  fewer  bacteria,  while  that  arising  from  the  large  intestine 
is  attended  with  the  presence  of  more,  as  the  contents  of 
the  large  intestine  exhibit  a  larger  number  of  microorgan- 
isms than  those  of  the  small  intestine.  Both  points  of  dis- 
tinction are,  however,  not  conclusive,  and  the  results  of 
bacteriologic  examination  of  the  peritonitic  exudate  can 
not  be  employed  without  reservation  as  a  certain  guide  for 
diagnostic  purposes,  nor  have  these  results  yet  acquired 
noteworthy  significance  from  a  prognostic  or  a  therapeutic 
point  of  view. 


154  CLINICAL  BACTERIOLOGY. 


PERITYPHLITIS. 

Perityphlitis,  like  other  inflammations  arising  from  the 
intestine,  is  due  to  the  activity  of  the  bacterium  coli  com- 
mune. Thus  far  only  perityphlitic  abscesses  and  their 
complications  have  been  submitted  to  examination,  coli- 
bacilli being  predominantly  found.  Actinomycotic  and 
tuberculous  perityphlitis  are  associated  with  the  presence 
of  their  special  exciting  agents. 

CHOLECYSTITIS   AND   CHOLANGITIS. 

Normally  the  bile  is  sterile,  the  bacterium  coli  and  cocci 
being  found  only  in  the  lowest  portion  of  the  choledoch 
duct.  Every  obstruction,  however,  that  interferes  with  the 
free  discharge  of  bile  (gall-stones,  etc.)  renders  possible  in- 
fection of  the  biliary  passages.  The  bacteria  present  in 
the  bowel,  particularly  the  bacterium  coli  commune,  less 
commonly  staphylococci,  streptococci,  and  pneumococci, 
gain  entrance  into  the  gall-bladder  and  the  biliary  passages, 
where  they  induce  inflammation  and  suppuration. 

Normal  bile  possesses  no  bactericidal  activity  ;  when  ob- 
tained in  a  sterile  condition,  it  constitutes  a  fairly  good 
nutrient  medium  for  coli-bacilli  and  staphylococci. 

Infection  of  the  unobstructed  biliary  passages  takes  place 
in  human  beings  in  individual  cases  of  typhoid  fever, 
cholera,  and  croupous  pneumonia.  It  may  be  assumed 
that  in  these  cases  also  the  infection  takes  place  through 
the  choledoch  duct.  Typhoid -bacilli  and  cholera-bacilli 
are  systematically  present  in  a  virulent  state  in  the  intestinal 
canal  throughout  the  course  of  the  respective  diseases  to 
which  they  give  rise.  The  pneumococcus  occurs  but 
seldom  in  the  intestinal  tract,  although  it  has  been  found 
there  with  certainty  in  individual  cases  of  pneumonia.  As 
a  result  of  the  constitutional  disease,  the  function  of  the 
liver  is  more  or  less  impaired,  the  excretion  of  bile  is  not 
normal,  and,  in  consequence,  infection  from  the  intestine  is 
possible. 

Infection  through  the  blood  is  probably  of  rare  occur- 
rence in  human  pathology.  In  experiments  on  animals 
this  may  be  attained  by  introducing  the  bacteria  in  large 
number,  or  by  previous  injury  of  the  biliary  passages — as, 
for  instance,  by  the  establishment  of  a  biliary  fistula. 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  155 

Experiments  on  Animals. — If,  after  ligation  of  the 
choledoch  duct,  coli-bacilli  are  introduced  in  the  central 
portion  of  the  duct,  the  animals  (dogs,  rabbits)  die  as  a 
result  of  purulent  cholecystitis  and  cholangitis.  If  the 
bacilli  are  injected  directly  into  the  mouth  of  the  choledoch 
duct  after  the  duodenum  has  been  opened,  the  same  result 
is  obtained  if  the  coli-cultures  are  sufficiently  virulent. 

The  bacteriologic  diagnosis  can  be  made  only  in  cases 
of  empyema  of  the  gall-bladder,  aseptic  puncture  being 
practised  and  plates  being  cast  with  the  bile  obtained.  Such 
puncture,  however,  should  not  be  undertaken  unless  urgently 
indicated,  as  even  when  made  with  most  careful  aseptic 
precautions,  signs  of  peritoneal  irritation,  usually  mild, 
follow. 


ABSCESS  OF  THE  LIVER. 

So-called  biliary  abscesses  belong  in  the  domain  of  cho- 
langitis. Pyemic  abscesses  of  the  liver  constitute  part  of  a 
purulent  general  infection.  (See  Pyemia  and  Its  Causes,  p. 
1 6 1.)  Hepatic  abscesses  in  the  sequence  of  gastro-intcs- 
tinal  lesions  arise  through  the  intermediation  of  the  portal 
vein  ;  coli-bacilli  act  as  exciting  agents.  With  regard  to 
tropical  abscesses  of  the  liver,  the  section  on  Dysentery  may 
be  consulted. 

In  the  bacteriologic  diagnosis  the  pus  obtained  on  ex- 
ploratory puncture  with  a  sterile  hypodermic  syringe  in 
the  search  for  the  abscess  is  subjected  to  examination. 


CYSTITIS. 

Numerous  investigations  in  recent  years  have  shown  that 
the  bacterium  coli  commune,  or  the  bacterium  aerogenes, 
plays  the  most  important  part  in  the  etiology  of  cystitis. 
The  other  microbes  of  suppuration  and  inflammation  also 
deserve  consideration  in  this  connection,  but  they  occur- 
less  frequently  than  the  aerogenes  and  the  coli.  An  excep- 
tion is  formed  by  puerperal  cystitis,  following  parturition, 
with  which  the  streptococcus  pyogenes  and  the  staphylo- 
coccus pyogenes  are  equally  associated  ;  also  by  gonorrheal 
cystitis,  which,  in  part  at  least,  is  dependent  upon  the  gono- 
coccus,  and  by  tuberculous  cystitis,  which  is  caused  by  the 
tubercle-bacill  u  s. 


156  CLINICAL  BACTERIOLOGY. 

The  bacteria  gain  entrance  into  the  urinary  bladder — 

1.  Through  unclean  instruments  employed  in  catheteri- 
zation. 

2.  By  ascending  through  the  urethra — this  is  especially 
the  case  in  women. 

3.  Through  the  intermediation  of  the  kidneys  ;  some 
bacteria  are  capable  of  passing  through  the  renal  filter;  and 
thus  gain  entrance  with  the  urine  into  the  bladder. 

Among  influences  favoring  the  development  of  cystitis 
are  cold,  traumatism,  and  retention  of  urine.  In  addition 
to  the  common  exciting  agents  of  suppuration  other  less 
common  microorganisms  have  been  found  in  individual 
cases  of  cystitis  :  the  so-called  micrococcus  albicans  am- 
pins, the  diplococcus  subflavus,  the  proteus,  and  others. 
These  bacteria  are,  however,  not  pathogenic  in  themselves, 
and  probably  acquire  importance  only  in  mixed  infection. 
The  proteus  causes  putrid  decomposition  of  the  urine. 

Bacteriologic  Diagnosis. — Catheterization  is  practised 
by  means  of  a  catheter  sterilized  by  boiling,  the  urine  being 
received  into  a  sterile  vessel.  Men  are  permitted  simply  to 
pass  urine  spontaneously,  the  last  portion  evacuated  being 
kept  for  examination,  and  the  orifice  of  the  urethra  having 
previously  been  thoroughly  cleansed.  From  the  urine  plate- 
cultures  are  rhade. 

Experiments  on  Animals. — By  injecting  pyogenic  cocci 
into  the  bladder  it  is  possible  to  induce  cystitis  in  male  ani- 
mals with  certainty,  if  the  escape  of  urine  is  prevented  for 
from  twelve  to  twenty-four  hours  by  ligation  of  the  penis. 

Ammoniacal  Fermentation  of  Urine. — The  bacterium 
coli  possesses,  in  only  slight  degree,  the  faculty  of  causing 
decomposition  of  urine.  Ammoniacal  fermentation  results 
from  the  action  of  individual  varieties  of  the  bacterium  coli 
in  the  presence  of  a  feebly  acid,  or  an  alkaline,  reaction  of 
the  urine.  Other  varieties  of  the  bacterium  coli  are  incap- 
able of  causing  decomposition  of  urine.  Ammoniacal 
cystitis,  however,  develops  in  the  presence  of  the  staphy- 
lococcus pyogenes  or  the  proteus,  whether  in  pure  culture 
or  in  mixed  infection  with  the  coli  commune. 

Pneumaturia. — In  some  cases  of  cystitis,  especially 
with  coincident  presence  of  sugar  in  the  urine,  but  also  in 
the  absence  of  glycosuria,  the  formation  of  gas  may  take 
place  in  the  bladder.  The  urine  under  such  circumstances 
is  evacuated  with   an  audible   sound  (pneumaturia).     This 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  157 

phenomenon  is  the  result,  in  some  cases,  of  fermentation  of 
the  sugar  in  the  urine  through  the  activity  of  microorgan- 
isms ;  in  other  cases  special  varieties  of  coli-bacilli  (aero- 
genes,  etc.),  characterized  by  abundant  production  of  gas, 
have  been  cultivated  from  such  urine.  In  one  such  case 
the  gases  formed  consisted,  in  addition  to  carbon  dioxid,  of 
free  nitrogen  and  hydrogen. 


NEPHRITIS. 

A  bacterial  origin  has  been  ascribed  to — 

1.  Primary  infectious  nephritis. 

2.  The  nephritis  occurring  as  a  complication  of  infec- 
tious diseases,  including  the  septic. 

Streptococci  have  repeatedly  been  found  in  association 
with  acute  nephritis,  and  both  in  the  urine,  from  which 
they  disappeared  with  the  termination  of  the  disease,  and 
after  death  in  the  kidneys,  in  which  they  were  visible  in  the 
vessels,  in  the  epithelium,  and  in  tube-casts.  Acute  nephri- 
tis has  been  induced  also  experimentally  in  animals  by 
injection  of  streptococci  into  the  blood.  Under  these  con- 
ditions a  large  number  of  streptococci  at  first  appeared  in 
the  urine,  but  these  disappeared  later.  The  disease,  how- 
ever, progressed  and  terminated  fatally,  but  no  cocci  were 
demonstrable  in  the  kidneys  after  death.  This  observation 
indicates  that  nephritis  may  be  of  bacterial  origin  without 
bacteria  being  found  in  the  kidneys  after  death  (Mannaberg). 
The  passage  of  the  bacteria  through  the  kidneys  may  suf- 
fice under  certain  circumstances  to  excite  the  anatomic  in- 
flammatory process,  which  may  then  pursue  its  further  course 
independently  of  them.  In  the  majority  of  cases  of  acute 
primary  nephritis  bacteria  are,  as  a  rule,  found  in  the  kid- 
neys. 

A  variety  of  inflammation  of  the  kidney  excited  by 
bacteria  appears  further  at  times  to  present  itself  from  the 
beginning  as  a  chronic  nephritis.  In  the  morbid  condition 
of  slow  course  induced  by  Gharrin  in  rabbits  by  inoculation 
with  pyocyaneus,  chronic  nephritis  was  found  repeatedly. 

In  some  cases  of  complicating  nephritis  the  exciting 
agents  of  the  primary  disease  have  been  present.  Thus, 
typhoid-bacilli  have  been  found  in  the  kidneys  in  associa- 
tion with  typhoid  nephritis  ;  diplococci,  in  association  with 
pneumonic  nephritis ;  and  spirilla  of  relapsing  fever,  in  the 


158  CLINICAL   BACTERIOLOGY. 

urine  of  a  patient  suffering  from  nephritis  complicating  that 
disease.  In  other  cases  streptococci  were  found  in  the  kidneys 
(smallpox,  rheumatism,  some  cases  of  scarlet  fever,  etc.)  ; 
under  these  conditions  it  is  possible  that  a  secondary  or  a 
mixed  infection  existed.  Finally,  complicating  nephritis 
need  not  be  excited  directly  by  the  bacteria,  but  it  may  be 
of  toxic  origin,  resulting  from  the  elimination  through  the 
kidneys  of  the  toxins  generated  by  the  bacteria  at  the  site 
of  the  primary  disease.  This  is  true  peculiarly  of  diphtheric 
nephritis  ;  in  conjunction  with  which,  likewise,  as  is  usual 
with  scarlatinal  nephritis,  bacteria  can  not  be  found  in  the 
majority  of  the  kidneys  examined. 

The  bacteriologic  diagnosis  can  be  made  during  life 
from  examination  of  the  urine  (possibly  after  centrifugation), 
when  there  is  no  disease  of  the  conducting  passages,  and 
especially  of  the  bladder.  Under  normal  conditions  the 
urine  that  enters  the  bladder  is  and  remains  sterile,  only 
becoming  contaminated  with  bacteria  in  the  urethra.  For 
this  reason  the  urine  removed  from  the  healthy  bladder 
with  a  sterile  catheter  may  be  transferred  directly  to  a 
nutrient  medium,  and  such  bacteria  as  develop  may  be  con- 
sidered as  being  derived  from  the  kidney.  In  men,  gener- 
ally, irrigation  of  the  urethra  with  the  first  half  of  the  urine 
contained  in  the  bladder  is  sufficient ;  the  last  half  is,  as  a 
rule,  sterile  in  healthy  individuals. 

Diagnostic  or  prognostic  significance  can  not  be  at- 
tached to  the  demonstration  of  bacteria  in  the  urine  in 
cases  of  nephritis.  According  to  Mannaberg,  streptococci 
are  found  in  the  urine  only  in  cases  of  true  acute  nephritis 
that  pursue  a  rapid  and  favorable  course,  while  they  are 
wanting  from  the  beginning  in  the  apparently  acute  cases 
that  subsequently  prove  to  be  chronic.  This  statement 
has,  however,  not  been  confirmed  by  other  observers. 


PERINEPHRITIS. 

Perinephritis  consequent  upon  disease  of  adjacent  organs 
(kidney,  intestine)  is  generally  due  to  the  bacterium  coli. 
Perinephritis  following  traumatism,  or  attending  general 
infection,  may  be  due  to  any  pyogenic  microorganism. 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  159 


PYELONEPHRITIS. 

A  distinction  is  to  be  made  between  an  ascending  and 
a  descending  pyelonephritis.  The  first  variety,  by  far  the 
more  common,  is  associated  with  the  presence  of  identi- 
cally the  same  microorganisms  as  is  cystitis,  upon  which  it 
is  dependent,  and  of  which  it  represents  the  final,  incurable 
stage.  The  microorganism  most  frequently  associated  is 
the  bacterium  coli  or  aerogenes.  The  descending  variety, 
with  infection  from  the  kidney,  is  usually  a  pyemic  process, 
and  is  dependent  upon  the  related  microorganism. 

In  ascending  pyelonephritis  the  bacteria  gain  entrance 
into  the  pelvis  of  the  kidney  as  the  result  of  retention  of 
urine.  No  longer  disturbed  by  the  discharge  of  urine, 
the  microorganisms  that  have  caused  the  inflammation  of 
the  bladder  wander  into  the  ureter  and,  multiplying  therein, 
they  finally,  by  extension  upward,  invade  the  pelvis  of  the 
kidney. 

Chronic  occlusion  of  the  ureter  may  be  followed  by  a 
pure  pyelonephritis,  without  preceding  cystitis,  when  the 
exciting  agents  of  inflammation  are  present  in  the  circulat- 
ing blood,  are  eliminated  through  the  kidneys,  and  collect 
in  the  stagnating  urine  in  the  pelvis  of  the  kidney. 

The  bacteriologic  diagnosis  is  only  possible  during  life 
if  operation  be  performed. 

Experiments  on  Animals. — After  ligation  of  the 
ureter,  pyelonephritis  can  be  induced  in  rabbits  by  means 
of  coli-bacilli  or  of  pyogenic  cocci,  both  by  injection  of 
these  bacteria  directly  into  the  pelvis  of  the  kidney  or  into 
the  ureter  above  the  ligature,  and  by  intravenous  iniection. 


INFLAMMATIONS  OF  THE  FEMALE   GENITAL  ORGANS. 

Vulvitis. — Suppurative  inflammation  of  the  vagina  is 
caused  by  the  exciting  agents  of  suppuration  ;  diphtheric 
inflammation,  by  the  diphtheria-bacillus ;  gonorrheal  in- 
flammation, by  the  gonococcus. 

Endometritis. — Puerperal  endometritis  is  always  of  bac- 
terial origin  (streptococci,  staphylococci,  coli-bacilli) ;  of 
the  remaining  varieties  of  endometritis  the  majority  are  to 
be  attributed  to  gonorrhea. 

Salpingitis  and  Oophoritis. — Inflammation  of  the  tubes 
and  ovaries  usually  represents  an  extension  of  the  endo- 


160  CLINICAL  BACTERIOLOGY. 

metritic  process.  The  bacteria  pass  by  continuity  from  the 
mucous  membrane  of  the  uterus  to  that  of  the  tube,  and 
from  this  to  the  ovary.  The  most  frequent  cause  is,  there- 
fore, gonorrhea.  In  addition,  the  presence  of  streptococci, 
staphylococci,  coH-bacilli,  and  Frankel's  pneumobaciUi  has 
been  demonstrated.  A  special  position  is  occupied  by 
tuberculosis  of  the  tubes.  Exceptionally,  the  oviducts  are 
attacked  by  actinomycosis.  The  oophoritis  that  occurs  in 
the  course  of  some  infectious  diseases  is  due  to  the  excit- 
ing agents  of  the  primary  disease,  or  they  are  caused 
secondarily  by  pyogenic  microorganisms. 

Perimetritis  and  Parametritis. — Primary  inflammation 
of  the  perimetrium  and  the  parametral  connective  tissue  is, 
almost  without  exception,  dependent  upon  puerperal  in- 
fection (streptococci,  staphylococci,  bacterium  coli,  in  mixed 
infection  also  proteus),  in  the  course  of  which  the  exciting 
agents  of  suppuration  gain  access  to  the  perimetrium  and 
the  pelvic  connective  tissue  by  way  of  the  lymph-channels. 
Secondary  inflammation  exhibits  the  same  etiologic  rela- 
tions as  salpingitis  and  oophoritis,  of  which  it  constitutes  a 
complication.  Gonorrhea  frequently  and  tuberculosis  less 
commonly  are  of  etiologic  significance  in  this  connection. 


INFLAMMATORY   DISEASES   OF  THE   EYE. 

Conjunctivitis. — The  following  microorganisms  have 
been  cultivated  in  cases  of  simple  conjunctivitis  :  Staphylo- 
cocci, streptococci,  pneumococci.  Diphtheric  conjunctivitis 
is  excited  by  the  diphtheria-bacillus,  gonorrheal  conjunc- 
tivitis by  the  gonococcus,  and  tuberculous  conjunctivitis 
by  the  tubercle-bacillus. 

Keratitis. — Some  cases  of  keratitis  owe  their  origin  to 
the  common  exciting  agents  of  inflammation  and  suppura- 
tion, which  at  times  gain  access  to  the  tissues  through  a 
lesion  of  the  cornea.  Hypopyon  originates  in  a  similar 
manner. 

Iritis  and  Choroiditis. — Inflammation  of  the  deeper 
structures  of  the  eye  arises  secondarily  by  extension  of  the 
pyogenic  organisms  from  the  cornea  (contact-infection),  or 
metastatically  through  dissemination  of  the  bacteria  by 
means  of  emboli  (puerperal  fever,  pyemia,  etc.).  The  same 
statement  applies  to  panophthalmitis.  Primary  inflamma- 
tion of  the  choroid  and  retina  is  usually  dependent  upon 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  161 

the  activity  of  the  metaboHc  products  of  bacteria,  which 
do  not  themselves  advance,  as  a  rule,  beyond  the  cornea. 

Sympathetic  Ophthalmia. — Deutschmann  observed  in 
cases  of  sympathetic  ophthalmia  an  infiltration  of  the  pia 
and  the  presence  of  phlogogenic  bacteria  in  the  optic-nerve 
sheath  of  the  sympathetically  affected  eye.  He  attributed 
the  sympathetic  inflammation  to  direct  invasion  of  the 
microbes  by  way  of  the  optic-nerve  path  from  the  primarily 
affected  eye.  These  observations  have,  however,  not  been 
confirmed,  and  the  parasitic  origin  of  sympathetic  ophthal- 
mia must  still  be  considered  as  undemonstrated. 

Chalazion  is  looked  upon  as  the  expression  of  a  chronic 
inflammation  of  the  tarsal  connective  tissue  that  is  caused 
by  the  entrance  of  the  exciting  agents  of  inflammation  into 
the  excretory  ducts  of  the  Meibomian  glands,  and  in  the 
hair-follicles  of  the  eyelashes.  Giant-cells  are  invariably 
present  in  the  granulation-tissue  of  the  chalazion,  and 
Tangl  observed  therein  the  presence  of  tubercle-bacilli. 
This  observation,  however,  has  not  been  repeated,  and 
numerous  inoculations  of  chalazion-tissue  upon  animals 
have  never  led  to  the  development  of  tuberculosis  in  these. 

Trachoma. — Diplococci  closely  resembling  gonococci 
have  been  repeatedly  demonstrated  in  the  contents  of  the 
trachoma-follicle.  The  transmission  of  the  disease  by 
means  of  these  microorganisms  appears  to  have  been  suc- 
cessful in  several  instances,  but  their  specific  significance  is, 
on  the  whole,  still  doubtful. 


PYEMIA   AND   SEPTICEMIA. 

Pyemia  and  septicemia  are  not  sharply  separable  from 
each  other  either  clinically  or  etiologically.  Both  are 
caused  by  the  exciting  agents  of  suppuration,  with  the 
difference,  however,  that  in  relation  with  septicemia  the 
element  of  intoxication  is  the  more  conspicuous ;  whereas, 
in  relation  with  pyemia  dissemination  of  the  bacteria  by 
way  of  the  blood-stream  and  development  of  multiple  foci 
of  suppuration  (metastases)  take  place.  So-called  putrid 
intoxication  and  acute  malignant  edema  (gangrene  fou- 
droyante  of  the  French)  must  not  be  considered  as  varie- 
ties of  ordinary  septicemia.  Both  of  these  disorders  occupy 
a  peculiar  position  from  the  etiologic  point  of  view,  as  the 
first  is  due  to  mixed  infection  with  putrefactive  bacteria, 


162  CLINICAL  BACTERIOLOGY. 

especially  the  proteus  of  Hauser  (see  Proteus-infections), 
and  the  second  is  dependent  upon  the  activity  of  a  special 
specific  cause — the  bacillus  of  malignant  edema  (vibrion 
septique,  see  Malignant  Edema).  In  connection  with  the 
pyemia  and  septicemia  of  human  beings,  therefore,  there 
are  to  be  considered  the  streptococcus  pyogenes,  the  staphy- 
lococcus pyogenes,  the  diplococcus  lanceolatus  Frankel, 
the  bacterium  coli  commune,  and  the  diplobacillus  pneu- 
moniae Friedlander.  The  starting-point  of  pyemia  and 
septicemia  is  usually  a  primary  focus  of  suppuration  or  of 
inflammation.  If  such  a  lesion  can  not  be  demonstrated, 
the  condition  is  designated  cryptogenetic  septicemia.  Under 
these  circumstances  the  primary  focus  has,  in  the  majority 
of  cases,  merely  escaped  detection  because  of  its  obscure 
situation.  Among  such  concealed  sources  of  septic  dis- 
orders may  be  mentioned  mediastinitis,  prostatic  abscesses, 
collections  of  pus  in  the  accessory  cavities  of  the  nose 
(antrum  of  Highmore,  ethmoid  sinuses,  etc.).  Only  in 
rare  cases  does  pyemia  or  septicemia  result  from  direct 
absorption  of  the  exciting  agents  of  inflammation  into  the 
blood  from  without  or  from  an  internal  surface  without  the 
formation  of  a  primary  focus. 

Bacteriologic  Diagnosis. — The  bacteria  can  by  no 
means  be  demonstrated  in  the  blood  in  all  of  the  cases. 
This  should  not  occasion  surprise  in  cases  of  septic  intoxica- 
tion,-as  the  microorganisms  are  present  in  the  blood  in 
small  number,  if  at  all.  In  cases  of  pyemia  the  bacteria 
can  be  more  frequently  demonstrated  in  the  blood,  espe- 
cially at  the  time  of  the  chills,  when  the  infected  thrombi 
are  set  free  and  give  rise  to  fresh  metastases ;  but  even 
here  the  results  of  blood-inoculation  are  often  negative. 

The  technic  for  examination  of  the  blood  is  simple. 
The  finger  of  the  patient  is  cleansed  by  means  of  soap, 
alcohol,  mercuric-chlorid  solution,  and  ether,  its  tip  is  slightly 
punctured  with  a  lancet  sterilized  in  the  flame,  and  the 
escaping  blood  is  smeared  upon  culture-media  by  means  of 
the  platinum  loop  heated  in  the  flame  ;  eventually,  plates 
are  made.  It  is  better,  because  of  the  small  number  of 
bacteria  circulating  in  the  blood  at  any  time,  to  employ  a 
considerable,  amount  of  blood.  This  is  aspirated  by 
means  of  a  sterilized  Roux  syringe  from  a  vein  distended 
by  compression. 

Metastatic  abscesses  are  opened  with  aseptic  precautions, 


OCCURRENCE  OF  CAUSATIVE  AGENTS.  163 

and  from  the  pus  cultures  are  made  directly,  or  plates  are 
poured  and  cover-slip  preparations  are  prepared. 

The  excretory  products  of  septic  patients — urine,  sweat, 
and  saliva — at  times  contain  the  exciting  agents  of  the  ex- 
isting disease,  and  in  suitable  cases  it  is,  therefore,  advisable 
to  examine  these  secretions  also.  The  elimination  of  the 
bacteria  through  these  channels  constitutes  a  mode  by 
which  the  organism  spontaneously  gets  rid  of  the  exciting 
agents  of  the  disease. 

Experiments  on  Animals. — Experimental  septicemia, 
such  as  may  be  induced  in  animals  by  means  of  the  most 
varied  microorganisms,  can  not  without  qualification  be 
considered  analogous  to  septicemia  in  human  beings.  In 
experiments  on  animals  unlimited  multiplication  of  the  bac- 
teria in  the  blood  takes  place,  although  it  is  to  be  borne  in 
mind  that  enormous  multiplication  sets  in  only  a  short 
time  before  the  death  of  the  animal.  The  conditions  are 
much  simpler  in  the  case  of  pyemia,  as  this  disorder  may 
be  induced  in  animals  through  all  portals  of  infection  and 
with  all  pyogenic  microorganisms.  It  is  a  necessary  con- 
dition, however,  that  the  microorganisms  employed  in  the 
experiment  shall  possess  a  sufficient  degree  of  virulence. 


PUERPERAL  FEVER. 

Puerperal  fever  is  only  clinically  a  special  form  of  pyemia 
or  septicemia  ;  but  in  relation  to  its  causative  agents,  analo- 
gous to  all  other  varieties.  The  streptococcus  pyogenes, 
the  staphylococcus  pyogenes,  and,  less  commonly,  the 
bacterium  coli  have  been  found  to  be  its  exciting  agents. 
The  severity  of  the  disorder  is  dependent  upon  the  fact 
that  the  bacteria  gain  entrance  directly  into  the  open  lumen 
of  the  vessels  of  the  uterine  mucous  membrane  injured  in 
the  process  of  parturition,  and  by  this  means  into  the  gen- 
eral circulation.  The  process  pursues  a  relatively  favor- 
able course  when  the  vessels  are  already  occluded  by 
thrombi,  and  the  larger  lymph-trunks  are  again  closed. 
The  bacteria  then  wander  through  the  lymph-spaces  be- 
tween the  muscle-fibrils,  reach  the  pelvic  connective  tissue, 
and  there  give  rise  to  a  localized,  circumscribed,  suppura- 
tive process,  so-called  puerperal  parametritis.  The  occur- 
rence simultaneously,  or  in  rapid  succession,  of  widely 
separated  foci  of  suppuration — as,  for  instance,  the  periton- 


164  CLINICAL   BACTERIOLOGY. 

itis,  the  empyema,  the  arthritis,  etc.,  of  puerperal  sepsis — 
can  only  take  place  through  the  dissemination  of  the 
germs  by  way  of  the  blood-stream. 

It  may  be  mentioned  that,  in  the  milk  of  nursing  women 
suffering  from  puerperal  fever,  pyogenic  cocci  can  not  rarely 
be  found. 

OSTEOMYELITIS. 

Osteomyelitis  is  not  a  specific  disease.  Staphylococci 
have  been  most  frequently  found  to  be  its  exciting  cause 
— both  the  aureus  and  the  albus  ;  less  commonly,  the 
streptococcus  pyogenes,  the  diplococcus  Frankel,  the 
typhoid-bacillus,  and  the  bacterium  coli  commune  have 
been  cultivated  from  osteomyelitic  foci.  In  accordance 
with  these  observations  osteomyelitis  may  be  considered 
as  a  form  of  pyemia  characterized  clinically  by  its  localiza- 
tion in  the  bone-marrow.  The  skin  and  the  open  cavities 
of  the  body  constitute  portals  of  entry  for  the  bacteria.  It 
is,  however,  by  no  means  necessary  that  primary  foci 
(furuncle,  panaris,  etc.)  should  exist  in  every  instance. 
The  forms  of  life  normally  present  upon  the  skin  and 
mucous  membranes  render  superfluous  such  an  assumption 
in  the  individual  case.  Osteomyelitis  occurs  exclusively 
in  young  individuals,  and  it  has,  therefore,  been  designated 
the  pyemia  of  the  developmental  period.  In  young  in- 
dividuals the  growing  zone  of  bone  represents  a  point  of 
lessened  resistance  at  which  bacteria,  when  they  gain 
entrance  into  the  circulation  from  any  source,  may  lodge 
and  multiply. 

Experimental  Evidence. — If  young  animals  (rabbits  or 
dogs)  are  inoculated  by  intravenous  injection  with  pyogenic 
microorganisms,  subperiosteal  abscesses  and  purulent  in- 
flammation of  the  medulla  of  bone  occur.  In  older  ani- 
mals similar  results  are  obtained  only  if  previously  a  frac- 
ture has  been  induced  ;  thus,  to  a  certain  degree,  a  point 
of  lessened  resistance  has  been  established  artificially. 
Under  such  conditions  osteomyelitic  changes  take  place  in 
the  situation  of  the  fracture.  These  experimental  obser- 
vations are  in  accord  with  the  facts  of  human  pathology — 
as  osteomyelitis,  as  has  been  mentioned,  is  a  disease  of 
early  life. 


OCCURRENCE  OF  CAUSATIVE   AGENTS.  165 


PYOCYANEOUS  GENERAL  INFECTION. 

The  bacillus  pyocyaneus  is,  in  general,  quite  a  harmless 
bacterium.  Its  presence  in  the  pus  of  wounds,  in  cases  of 
otitis  media,  etc.,  retards  the  process  of  healing  in  the  re- 
spective diseases  only  inconsiderably,  and  gives  rise  to  the 
well-known  greenish  or  bluish  discoloration  of  the  pus 
and  the  dressings.  In  the  bodies  of  children  the  bacillus 
pyocyaneus  at  times  exhibits  pernicious  activity,  and  it 
appears  under  some  circumstances  capable  of  causing 
severe  general  infection.  Neumann  obtained  this  micro- 
organism from  the  blood  and  the  internal  organs  of  a  new- 
born infant  dead  of  hemorrhagic  septicemia.  H.  Kossel 
found  it  in  children  in  the  meningeal  exudation  in  the 
sequence  of  otitis,  in  diarrheal  stools,  in  cases  of  nephritis, 
and  in  the  presence  of  inflammatory  affections  of  the  naso- 
pharyngeal space.  He  believes  that  the  bacillus  is  capable 
of  causing  serious  injury  to  the  organism  of  the  child, 
either  directly  through  the  intermediation  of  the  blood- 
stream or  indirectly  through  its  metabolic  products. 


PART  III. 


SPECIFIC  DISEASES  OF  BACTERIAL  ORIGIN. 

TYPHOID  FEVER, 

Morphology  of  the  Typhoid-bacillus. — The  bacillus 
of  typhoid  fever  was  first  observed  by  Koch  and  Eberth^ 
and  grown  in  pure  culture  by  Gaffky  in  1884. 


Fig.  48.— Bacillus  typhi,  from  an  agar-agar  culture  twenty-four  hours  old ;  X  650 

(Heim). 

The  typhoid-bacilli  are  small,  plump  rods,  with  rounded  ex- 
tremities (0.5-0.9  X  1-3  /^)-  In  the  tissues  the  organisms 
lie,  as  a  rule,  isolated,  while  in  cultures  they  are  arranged  in 
pairs  and  not  rarely  in  long  filaments.  They  possess  from  eight 
to  eighteen  terminal  and  lateral  flagella,  and,  in  consequence, 
they  are  most  actively  motile,  their  movement  being  serpentine 
in  character.  They  do  not  stain  so  well  as  other  bacteria  and  they 
take  the  stain  with  some  difficulty.  It  is,  therefore,  advisable  to 
warm  the  aqueous  staining  solutions  and  also  the  diluted  carbol- 
fuchsin  solution.     The  bacilli  do  not  stain  by  Gram's  method. 

166 


TYPHOID   FEVER.  167 

Spore-formation. — Gaffky  considered  as  spores  certain  ter- 
minal bright,  oval  bodies  that  are  said  to  remain  unstained.  These 
bodies  (polar  granules)  have,  in  accordance  with  subsequent  in- 
vestigations, come  to  be  looked  upon  as  involution-forms.  At 
any  rate  the  bacilli  that  contain  such  bodies  are  not  noted  espe- 
cially for  their  powers  of  resistance,  as  they  are  destroyed  with 
certainty  by  exposure  for  ten  minutes  to  a  temperature  of  60°  C. 
(140°  F.). 

The  typhoid-bacillus  grows  in  the  absence  of  oxygen,  though 
by  no  means  so  well  as  in  its  presence  (facultative  anaerobiosis). 
The  temperature-optimum  for  the  typhoid-bacillus  is  that  of  the 
body.  The  organism  thrives  well,  however,  at  room-tempera- 
ture.    The  temperature-maximum  is  46°  C.  (114.8°  F.). 

The  Appearance  of  Typhoid-bacilli  in  Cultures. — 
The  bacillus  of  typhoid  fever — in  contrast  with  most  other  patho- 
genic bacteria — exhibits  vigorous  growth  on  slightly  acid 
nutrient  media. 

On  gelatin-plates  deep  colonies  appear  as  small,  punctate, 
sharply  circumscribed  dots ;  with  low  powers  of  the  microscope 
they  may  exhibit  a  brownish-yellow  color  and  a  whetstone 
shape.  Superficial  colonies  are  much  larger,  and  form  a  bluish, 
iridescent,  delicate  coating,  with  an  irregularly  serrated  border. 
Only  the  central  portion  of  the  colony  appears,  with  low  powers 
of  the  microscope,  of  a  yellowish  color,  while  toward  the  margin 
a  delicate  linear  network  can  be  observed,  giving  rise  to  a  leaf- 
like appearance.     The  gelatin  is  not  liquefied. 

In  gelatin  stab-cultures  development  takes  place  along  the 
entire  line  of  inoculation.  Superficial  growth  is  pronounced, 
and  presents  characteristics  similar  to  those  observed  in  superfi- 
cial colonies  on  plates. 

Gelatin  Streak-cultures. — From  the  center  the  entire  surface 
of  thfe  gelatin  is  covered  by  a  delicate,  iridescent,  bluish  coating. 

In  all  gelatin-cultures  a  peculiar  milky  turbidity  of  the  nu- 
trient medium  frequently  occurs  in  the  neighborhood  of  the 
culture. 

In  agar  streak-cultures  and  in  blood-serum  a  whitish  coating 
of  inconsiderable  density  forms,  without  characteristic  peculiari- 
ties. 

The  appearance  oi potato-cultures  is  of  importance.  In  these 
the  typhoid-bacillus  grows  in.  an  invisible  layer,  with  an  ap- 
pearance as  if  nothing  at  all  had  developed  upon  the  surface 
of  the  potato.  If,  however,  an  attempt  is  made  to  remove 
material  with  a  platinum  loop,  it  is  at  once  found  that  the  potato 
is  entirely  covered  by  a  layer  of  some  kind.  Microscopic  ex- 
amination confirms  this  observation,  and  discloses  the  presence 
of  large  numbers  of  actively  motile  rods.  This  mode  of  devel- 
opment is  quite  peculiar,  and  occurs,  so  far  as  is  as  yet  known. 


leS  CLINICAL  BACTERIOLOGY. 

only  with  typhoid-bacilli.  It  is,  however,  not  constant.  There 
are  varieties  of  potato  upon  which  the  bacilli  of  typhoid  fever 
develop  in  yellowish  or  brownish,  raised,  and  sharply  circum- 
scribed deposits,  and  generally  upon  potatoes  whose  surface 
yields  a  neutral  or  even  an  alkaline  reaction.  Such  visible 
growth  can  be  obtained  also  artificially  by  rendering  the  surface 
to  be  inoculated  of  alkaline  reaction.  Typical  characteristic 
growth  takes  place  only  when  the  potatoes  exhibit  an  acid  reac- 
tion, which  they  do,  as  a  rule. 

In  milk  the  typhoid-bacillus  induces  slight  formation  of  acid, 
but  never  coagulation.  In  Petruschky's  whey  (p.  Z(i)  the 
typhoid-bacillus  generates  not  more  than  three  per  cent,  of  acid, 
whereas  the  bacterium  coli  commune  generates  more  than  seven 
per  cent. 

Bouillon  is  rendered  turbid  by  cultures  of  the  typhoid-bacillus. 

The  typhoid-bacillus  does  not  induce  fermentation  in  nutrient 
media  containing  grape-sugar,  milk-sugar,  or  cane-sugar ;  nor 
does  it  form  indol  in  a  solution  of  peptone  and  sodium  chlorid. 

If  potassium  nitrite  and  sulphuric  acid  are  added  to  peptone- 
cultures,  a  red  coloration  does  not  occur  (p.  122,  bacterium 
coli  commune). 

The  typhoid-bacillus,  as  well  as  the  bacterium  coli  commune 
and  the  entire  group  of  bacteria  resembling  both,  is  character- 
ized by  a  certain  degree  of  resistance  to  carbolic  acid,  addition 
of  which  to  the  nutrient  medium  in  the  proportion  of  %  per 
cent,  not  inhibiting  the  bacilli  in  their  growth. 

Vital  Activity  of  the  Typhoid-bacillus. — Typhoid- 
bacilli  retain  their  vitality  in  sterilized  water  iox  a  considerable 
time  (up  to  three  months)  ;  they  may  even,  at  least  at  first, 
multiply  therein.  In  unsterilized  water  they  die  in  the 
course  of  two  weeks,  in  consequence  of  the  activity  of  the 
water-bacteria,  by  which  they  are  suppressed,  and  more 
quickly  in  running  water  than  in  stagnant  water.  Under 
favorable  conditions,  protected  from  light,  evaporation,  and 
competition,  they  may  persist  for  a  long  time.  Milk  may 
at  times  contain  living  typhoid-bacilli  for  as  long  a  period 
as  five  weeks.  In  the  slime  of  streams  and  of  wells  typhoid- 
bacilli  retain  their  capability  of  development  for  not  less 
than  three  weeks.  Buried  in  the  superficial  layers  of  the 
earth,  they  have  been  demonstrated  in  a  living  state  after 
five  and  a  half  months.  They  appear  also  capable  of  per- 
sisting for  an  equal  length  of  time  in  the  feces — for  three 
months  and  more  ;  naturally  only  when  too  many  putrefac- 
tive bacteria  are  not  present  at  the  same  time.     Typhoid- 


TYPHOID   FEVER.  169 

bacilli  bear  cold  quite  well ;  they  are  not  injured  by  freez- 
ing for  two  or  three  times,  and  subsequent  thawing.  They 
are  less  resistant  to  heat^  as  has  been  mentioned.  (See 
Spore-formation.) 

Dried  in  a  thin  layer,  typhoid-bacilli  have  been  found  to 
preserve  their  vital  activity  (Uffelmann) — 

In  garden-soil  for  twenty-one  days. 

In  sweepings  for  more  than  thirty  days. 

In  white  filter-sand  for  eighty-two  days. 

Upon  linen  for  from  sixty  to  seventy-two  days. 

Upon  buckskin  for  from  eighty  to  eighty-five  days. 

Upon  wood  for  thirty-two  days. 

According  to  Kruse,  they  die  in  thin  layers,  dried,  with- 
in from  five  to  fifteen  days. 

Portals  of  Infection  and  Dissemination  of  Typhoid- 
bacilli. — Dried  typhoid-germs  may  be  carried  through  the 
air  with  floor-dust,  street-sweepings,  particles  of  clothing, 
etc.  They  may,  thus,  possibly  be  inhaled.  Infection  by 
way  of  the  lungs,  however,  is  rather  improbable  with  regard 
to  typhoid  fever,  although  it  played  an  important  part 
in  the  earlier  theories  that  denied  the  transmission  from 
case  to  case,  and  considered  necessary  the  presence  of  the 
typhoid-bacilli  in  the  earth  for  their  complete  maturation ;  but 
this  has  not  been  demonstrated.  Besides,  in  human  beings 
the  digestive  tract  appears  to  be  the  only  portal  of  entry  for 
the  typhoid-bacillus.  The  typhoid-germs  must  be  swal- 
lowed, and  gain  entrance  into  the  intestine.  To  this  end 
it  is  necessary  that  they  lodge  upon,  and  be  taken  up  with, 
food.  Apparent  infection  through  the  lungs  may  also  be 
so  interpreted  that  the  typhoid-germs  contained  in  dust 
and  with  this  inhaled  are  restrained  in  the  upper  part  of 
the  respiratory  tract,  to  be  subsequently  carried  with  food 
into  the  digestive  tract.  It  is  an  important  fact  that  the 
typhoid-bacillus  is  not  destroyed  with  certainty  by  the 
hydrochloric  acid  of  the  gastric  juice.  The  barrier  inter- 
posed by  the  stomach  thus  fails  to  afford  reliable  protection 
against  typhoid  infection,  even  when  the  function  of  that 
organ  is  completely  normal. 

Articles  of  food  may  be  contaminated  with  typhoid-bacilli 
through  the  intermediation  of  the  air ;  dust  containing 
typhoid-bacilli  may  be  deposited  upon  articles  of  food. 
More  frequent,  however,  is  direct  contamination  by  means 
of  the  feces,  which  become  attached  to  the  hands  of  the  at- 


170  CLINICAL  BACTERIOLOGY. 

tendants  in  the  emptying  of  bed-pans,  and  in  the  cleansing 
of  soiled  linen,  and  transference  to  food  from  want  of  clean- 
liness. In  this  way  infection  from  case  to  case  takes  place, 
as  may  often  enough  be  demonstrated.  The  disease  oc- 
curs in  epidemic  distribution  when  a  common  article  of  food 
is  contaminated.  Thus,  epidemics  of  typhoid  fever  have 
been  caused  by  milk,  by  oysters  obtained  from  infected 
water,  and  more  frequently  by  drinking-water.  Repeatedly, 
a  communication  between  wells  or  sources  of  water-supply 
and  neighboring  cesspools  into  which  undisinfected  stools 
have  been  emptied  has  been  found  to  be  the  cause  of 
typhoid  epidemics.  In  other  epidemics  contamination  of 
the  pubHc  water-supply  by  means  of  typhoid  dejections,  or 
through  the  washing  of  infected  clothes,  has  most  proba- 
bly taken  place. 

Recently,  typhoijd-bacilli  have  been  observed  a  number 
of  times  without  a  direct  relation  to  cases  of  typhoid  fever 
being  demonstrable  or  even  suspected.  Losener  found 
bacilli  that  corresponded  in  all  respects  with  Eberth-Gafif  ky 
bacilli  in  a  specimen  of  earth  obtained  from  an  untilled 
field,  and,  further,  in  the  water-supply  of  Berlin  obtained 
from  a  conduit  in  his  laboratory.  Remlinger  and  Schneider, 
using  perfect  methods,  have  recently  cultivated  the  same 
microorganisms  from  earth,  dust,  and  water.  They  even 
found  them  in  the  intestines  of  five  individuals  who  had 
never  suffered  from  typhoid  fever.  Observations  like  these 
are,  however,  exceptional,  and  are  as  yet  beyond  explana- 
tion. 

In  order  that  typhoid  fever  may  develop,  in  addition  to 
the  taking  up  of  the  bacilli  a  special  predisposition  on  the 
part  of  the  individual  certainly  appears  necessary  ;  or  the 
bacilli  must  possess  especial  virulence  or  be  taken  up  in 
excessive  number.  In  general,  human  beings  may  be  con- 
sidered as  not  particularly  susceptible  to  typhoid  fever.  The 
requirement  of  an  especial  temporal  and  local  predisposition 
(elevation  of  the  ground- water),  as  demanded  by  the  theory 
that  the  condition  of  the  soil  bears  some  relation  to 
the  occurrence  of  typhoid  fever,  can  no  longer  be  sustained. 

The  Occurrence  of  the  Bacilli  in  Typhoid  Patients. — 
The  bacilli  are  found  systematically  in  Peyer  s  patches,  the 
mesenteric  glands,  the  spleen,  the  liver,  and  the  bone-marrow 
of  typhoid  patients.  They  are  always  collected  in  groups 
that  frequently  exhibit  a  relation  with  blood-vessels.     In 


TYPHOID   FEVER.  171 

the  feces  they  are  not  found  before  the  second  week 
of  the  disease,  usually  after  the  tenth  day,  but,  as  a  rule, 
not  in  large  number.  In  most  cases  they  disappear  from 
the  stools  as  early  as  the  fourth  week  of  the  disease,  and 
in  rare  cases  they  are  demonstrable  until  after  defervescence. 
The  blood  of  typhoid  patients  is  generally  sterile,  although 
typhoid-bacilli  have  been  found  in  the  blood  of  the  rose- 
spots  and  from  the  veins.  It  is  necessary  to  employ  con- 
siderable amounts  of  blood  (i  cu.  cm.)  for  the  purpose  of 
examination.  The  urine  during  life  and  the  kidneys  after 
death  frequently  contain  the  bacilli,  especially  in  cases 
complicated  by  albuminuria.  Rarely  the  bacilli  have  been 
found,  beside's,  in  the  hings  in  some  cases  of  typhoid  pneu- 
monia or  bronchopneumonia,  in  the  meninges  in  cases  of 
typhoid  meningitis,  in  the  myocardium  in  cases  of  typhoid 
myocarditis,  and  in  the  testicles  in  cases  of  typhoid  orchitis. 

Typhoid-bacilli  have  been  found  repeatedly  in  the  pus  of 
suppurative  processes  that  develop  in  the  course,  or  as  a 
sequel,  of  an  attack  of  typhoid  fever :  as,  for  instance, 
osteoperiostitic  processes,  encapsulated  peritonitis,  abscesses 
of  the  spleen  and  the  liver,  inflammation  of  joints,  thyroiditis, 
empyema,  etc.  These  observations  show  that  the  typhoid- 
bacillus  is  also  capable  of  manifesting  pyogenic  activity. 
Typhoid-bacilli  have  been  found  in  such  posttyphoid  sup- 
purative processes  fifteen  months  after  the  termination  of 
the  disease,  and  in  one  case  of  old  bone-disease  as  long  as 
seven  years  afterward.* 

Mixed  Infection  and  Secondary  Infection. — The  course 
of  typhoid  fever  is  frequently  modified  by  complicating  in- 
fections due  to  other  microorganisms,  usually  those  exciting 
inflammation.  At  times  these  complications  appear  at  the 
commencement  of  the  disease  as  the  expression  of  a  mixed 
infection  ;  at  other  times  they  occur  later  as  secondary  in- 
fections. The  bacterium  coli  commune  plays  the  most  im- 
portant part  in  the  development  of  these  conditions.  It  is 
found  to  be  the  cause  of  peritonitis,  cholangitis,  etc.  Next 
in  importance  is  the  streptococcus  pyogenes,  which  has 
been  frequently  demonstrated  in  cases  presenting  secondary 
empyema,  otitis,  and  bronchopneumonia.      Mixed  infection 

*Hunner,  "Bulletin  of  the  Johns  Hopkins  Hospital,"  Aug.-Sept.,  1899, 
p.  163,  has  reported  a  case  of  acute  suppurative  cholecystitis  in  which  typhoid- 
bacilli  were  isolated  from  the  contents  of  the  gall-bladder  eighteen  years  after 
an  attack  of  typhoid  fever. — A.  A.  E. 


172  CLINICAL  BACTERIOLOGY. 

with  streptococci  is  responsible  for  the  development  of  the 
dangerous  condition  known  as  streptococcous  typhoid  septi- 
cemia. The  investigations  of  Vincent  have  shown  that  the 
presence  of  the  streptococcus  and  artificial  mixed  cultures 
materially  increases  the  virulence  of  typhoid-bacilli.  The 
staphylococcus  pyogenes,  the  diplococcus  lanceolatus 
Frankel,  and  the  proteus,  likewise  frequently  find  oppor- 
tunity to  gain  lodgment  in  the  organism  enfeebled  by 
typhoid  fever,  and  to  give  rise  to  the  development  of  fur- 
uncles, cutaneous  abscesses,  bronchopneumonia,  catarrh  of 
the  middle  ear,  gangrene  of  the  skin,  and  the  like. 

Experiments  on  Animals, — Mice,  guinea-pigs,  rabbits, 
goats,  etc.,  die  after  the  introduction  of  virulent  Eberth- 
Gaff  ky  bacilli,  with  decline  of  temperature,  convulsions,  and 
diarrhea.  Considerable  numbers  of  bacteria  are  necessary 
in  subcutaneous  introduction,  while  smaller  numbers  suffice 
in  intraperitoneal  and  intravenous  inoculation.  The  fatal 
result  is  brought  about  under  these  conditions  through 
intoxication.  If  the  virulence  of  the  infectious  agents  is 
marked,  the  animals,  and  especially  white  mice,  may,  how- 
ever, be  destroyed  by  intraperitoneal  injection  of  quite 
small  numbers  of  bacteria,  although  large  numbers  of  the 
bacilli  are  found  in  the  blood  after  death.  It  is  certain 
that  under  these  conditions  an  increase  has  taken  place  in 
the  number  of  bacteria,  and  it  can,  therefore,  not  be  denied 
that  the  disease  induced  in  animals  by  typhoid-bacilli  dis- 
plays the  characters  of  a  true  infection. 

The  development  of  actual  typhoid  fever  in  animals  (and 
also  mere  typhoid-bacilli  intoxication)  in  consequence  of 
introduction  of  the  bacteria  through  the  mouth  is  attended 
with  serious  difficulties.  It  is,  however,  to  be  observed 
that  a  true  typhoid  disease  appears  not  to  exist  naturally  in 
animals.  If  preliminary  treatment  is  employed  similar  to 
that  adopted  in  the  experimental  development  of  cholera — 
alkalinization  of  the  gastric  contents,  and  injection  of  tinc- 
ture of  opium — changes  are  at  times  induced  in  guinea-pigs 
by  the  introduction  of  typhoid-bacilli  through  the  mouth  that 
in  some  degree  are  suggestive  of  those  of  typhoid  fever  in 
human  beings.  Experiments  on  animals  of  a  completely 
positive  character,  with  the  pathologic-anatomic  lesions  of 
true  typhoid  fever,  have  not  been  recorded  in  the  literature. 

The  pyogenic  activity  of  the  typhoid-bacillus  can  be 
readily  demonstrated  in  experiments  on  animals. 


TYPHOID   FEVER.  173 

Etiologic  Relations  of  the  Bacilli  to  Typhoid  Fever 
in  Human  Beings. — In  view  of  their  constant  presence  in 
all  cases  of  typhoid  fever,  and  of  their  occurrence  exclu- 
sively in  this  disease,  the  Eberth-Gaffky  bacilli  may  be 
considered  as  the  exciting  agents  of  typhoid  fever,  although 
experimental  development  of  typhoid  fever  by  means  of  the 
bacilli  has  not  been  induced.  The  bacteria  that  gain 
entrance  into  the  digestive  tract  with  the  food  obtain  lodg- 
ment, in  the  presence  of  the  necessary  predisposition  on  the 
part  of  the  individual,  in  the  follicles  and  plaques  of  the 
intestinal  wall.  The  period  of  incubation  for  typhoid  fever 
is  from  one  to  three  weeks.  During  this  time  the  bacteria 
slowly  give  rise  to  the  anatomic  process  that  constitutes  the 
basis  of  the  disease,  the  swelling  of  the  plaques,  and,  later, 
their  ulceration.  At  the  same  time  the  bacteria  multiply 
and  gain  entrance — probably  by  way  of  the  lymph-paths 
especially — into  the  mesenteric  glands,  also  into  the  liver 
and  the  spleen.  To  this  point  typhoid  fever  is  a  truly  in- 
fectious disease.  The  toxic  activity  of  the  bacteria  is,  how- 
ever, of  equally  marked  importance.  Brieger  and  Frankel 
have  demonstrated  in  bouillon-cultures  of  typhoid-bacilli  a 
chemic  poison  that  they  place  in  the  class  of  toxalbumins. 
R.  Pfeiffer  also  held  the  view  with  regard  to  typhoid  fever 
that  the  peculiar  poison  resides  in  the  bodies  of  the  bacteria. 
(See  General  Section,  p.  31.)  This  can  be  readily  obtained 
by  destroying  young  agar-cultures  carefully  by  means  of 
chloroform-vapor  or  by  exposure  for  an  hour  to  a  tempera- 
ture of  54°  C.  (129.2°  F.).  From  eight  to  ten  mg.  of  the 
bacterial  mass  suffice  to  destroy  a  guinea-pig.  There  is  no 
doubt  that  also  in  the  body  of  the  typhoid  patient  a  specific 
poison  is  generated  by  the  bacteria,  and  is  absorbed.  The 
characteristic  mental  dulness,  and  the  peculiar  febrile  course 
may  be  considered  as  expressions  of  this  intoxication.  Not 
rarely  cases  of  typhoid  fever  are  observed  in  which  the  intes- 
tinal lesions  are  subordinate  to  the  toxic  state.  These  are 
instances  of  typhoid  fever  with  most  profound  mental  con- 
fusion and  high  fever,  but  with  mild  intestinal  manifestations, 
in  which  after  death  only  a  few  ulcers  of  small  extent  are 
found  in  the  bowel.  Cases  have  even  been  reported  in  which 
during  life  the  characteristic  stools,  and  after  death  the 
typhoid  ulcers,  were  entirely  wanting  :  the  diagnosis  of 
typhoid  fever  being  based  solely  upon  the  demonstration  at 
the  autopsy  of  typhoid-bacilli  in  the  greatly  enlarged  spleen. 


174  CLINICAL  BACTERIOLOGY. 

Bacteriologic  Diagnosis  of  Typhoid  Fever  (Differentia- 
tion of  Typhoid-bacilli  and  Bacterium  Coli  Commune). 

— The  bacteriologic  diagnosis  of  typhoid  fever  is  usually 
complicated  by  so  many  difficulties  that  it  can  not  be  con- 
sidered as  an  aid  in  the  clinical  diagnosis.  Isolation  of  the 
typhoid-bacillus  in  the  feces  is  a  matter  of  great  difficulty  by 
reason  of  the  extraordinary  resemblance  of  the  bacillus  to  the 
bacterium  coli  commune — a  resemblance  that  is  so  marked 
that  some  observers  (particularly  the  Lyons  school)  have 
considered  the  two  microorganisms  identical.  The  micro- 
scopic appearances,  the  agar-culture  and  the  gelatin-culture 
of  both  bacteria  are  absolutely  alike.  Potato-cultures 
are,  generally,  though  by  no  means  always,  different :  the 
bacterium  coli  gives  rise  to  a  thick,  raised,  circumscribed, 
greasy,  brownish  deposit  ;  the  typhoid-bacillus,  on  the 
other  hand,  to  an  invisible,  extensive  coating.  In  using 
potatoes  for  differential  diagnostic  purposes  one-half  of  the 
surface  must  be  inoculated  with  the  suspected  bacteria,  and 
the  other  half  with  actual  typhoid-bacilli.  If  the  same 
growth  takes  place  on  each  half  of  the  potato,  it  is  highly 
probable,  providing  there  is  agreement  in  other  respects, 
that  the  suspected  organism  is  really  the  specific  Eberth- 
Gafifky  bacillus.  Among  further  points  of  differential 
diagnosis  as  between  typhoid-bacilli  and  coli-bacilli  are  the 
following:  (i)  The  bacterium  coli  coagulates  milk;  the 
typhoid-bacillus  does  not.  (2)  The  bacterium  coli  gener- 
ates gas  in  nutrient  media  containing  peptone,  and  especi- 
ally in  those  containing  grape-sugar,  after  exposure  in 
the  thermostat  for  even  a  few  hours  ;  the  typhoid-bacillus 
does  not.  It  must,  however,  not  be  overlooked  that, 
though  quite  seldom,  there  occur  varieties  of  coli-bacilli 
that  do  not  coagulate  milk  and  do  not  cause  fermentation 
of  grape-sugar,  and  thus  can  not,  by  any  of  the  methods 
thus  far  known,  be  absolutely  differentiated  from  the 
Eberth-Gaffky  bacillus.  A  further  point  of  differentiation 
consists  in  the  fact  that  the  typhoid-bacillus  does  not  yield  the 
indol-reaction,  whereas  the  bacterium  coli  does.  There  is, 
however,  a  variety  of  bacterium  coli  that  does  not  generate 
indol.  The  differentiation  between  the  two  bacteria  thus  re- 
mains extremely  difficult,  and  only  such  bacteria  can  be 
decided  with  perfect  certainty  to  be  typhoid-bacilli  as 
possess  all  of  the  peculiarities  described,  and  as  have 
been    cultivated   from   the   spleen   of  human    beings  who 


TYPHOID   FEVER.  175 

exhibit,  or  have  exhibited,  the  dinical  symptoms  of  typhoid 
fever. 

Recent  and  reliable  aids  in  the  differential  diagnosis 
between  the  bacillus  of  Eberth-Gaff  ky  and  the  great  horde 
of  bacteria  resembling  the  typhoid-bacillus  and  the  bac- 
terium coli  are  furnished  by  the  reaction  of  Pfeiffer  and 
that  of  Gruber.  (See  General  Section,  pp.  62  and  63.) 
Ten  times  the  minimal  lethal  dose  amount  of  the  sus- 
pected bacteria  are  mixed  with  a  small  amount — less  than 
o.  I  cu.  cm. — of  serum  obtained  from  an  animal  highly  im- 
munized against  typhoid  fever,  and  the  mixture  is  injected 
into  the  peritoneum  of  a  guinea-pig.  If  after  from  ten  to 
twenty  minutes  the  characteristic  disintegration  of  the  bac- 
teria into  granules  becomes  apparent  in  the  fluid  obtained 
from  the  abdominal  cavity  by  means  of  a  capillary  tube, 
the  conclusion  may  be  reached  that  the  organisms  are 
actually  typhoid-bacilli.  If  the  virulence  of  the  microor- 
ganisms is  slight  or  wanting,  Pfeiffer' s  reaction  is  obviously 
not  available.  Under  these  conditions  Gruber' s  reaction  is 
employed.  The  technic  has  been  described  in  the  general 
section  (p.  64).  If  agglutination  takes  place,  the  diagnosis 
may  be  considered  as  positive. 

Typhoid-bacilli  are  isolable  from  the  feces  only  with 
great  difficulty  on  account  of  their  close  resemblance  to 
the  bacterium  coli  commune.  The  procedure  most  likely 
to  prove  successful  is  that  recommended  by  Eisner. 
Plates  are  made  in  the  usual  manner  with  potato-gelatin 
(see  Methods  of  Culture  and  of  Examination,  p.  81),  to  which 
shortly  before  use  potassium  iodid  has  been  added  in  the  pro- 
portion of  one  per  cent.  This  culture-medium  appears  espe- 
cially adapted  for  the  growth  of  typhoid-bacilli  and  coli-bac- 
teria,  with  the  qualification  that  the  latter  grow  more  vigor- 
ously than  the  former.  After  the  lapse  of  forty-eight 
hours  the  colonies  of  coli-bacilli  appear  as  dark -brown 
spherical  masses,  while  those  of  the  typhoid-bacilli  appear 
as  small,  transparent,  water-like  drops.  The  method  of 
Eisner  is,  however,  not  absolutely  trustworthy.  Its  appli- 
cation requires  much  practice,  and  it  does  not  render 
superfluous  further  precise  identification  of  the  apparent 
typhoid-colonies  according  to  all  of  the  rules  mentioned. 

The  bacteriologic  diagnosis  of  typhoid  fever  may  be 
made  quickly  and  easily  by  puncture  of  the  spleen,  and  the 
development   of  cultures  from  the  fluid  obtained.     If  the 


176  CLINICAL  BACTERIOLOGY. 

disease  present  is  typhoid  fever,  pure  cultures  may  thus  be 
obtained  at  once,  which  do  not  coagulate  milk  and  do  not 
generate  gas  in  peptone-bouillon.  In  rare  cases  of  mixed 
infection,  in  addition  to  typhoid-bacilli  the  streptococcus 
pyogenes  or  the  staphylococcus  is  also  found  in  the  fluid 
from  the  spleen.  The  method  of  puncturing  the  spleen  is 
the  same  as  that  employed  in  every  other  form  of  explora- 
tory puncture.  On  aspiration  the  splenic  fluid,  mixed  with 
blood,  is  readily  obtained,  and  this  is  injected  into  a  sterilized 
dish,  five  agar-tubes  being  each  then  smeared  successively 
with  a  drop  of  the  fluid  ;  with  the  remainder  plates  are  made, 
and  milk-flasks  and  peptone-bouillon  tubes  are  inoculated. 
Notwithstanding  the  trustworthy  character  of  its  results, 
puncture  of  the  spleen  is,  however,  not  to  be  recom- 
mended. The  procedure  is  by  no  means  free  from  danger. 
It  should  not  be  forgotten  that  the  typhoid-bacillus  is  also 
capable  of  pyogenic  activity,  and  that  the  puncture-track 
in  the  spleen  may  give  rise  to  purulent  complications, 
possibly  in  the  peritoneum. 

The  most  valuable  service  in  the  clinical  diagnosis  of 
typhoid  fever  appears  to  be  rendered,  however,  by  the  pro- 
cedure of  Widal,  who,  as  has  already  been  mentioned 
(p.  66),  showed  that  also  the  blood-serum  of  typhoid 
patients  yields  the  agghitinatioii-phenomenon  of  Gruber. 
Blood-serum  from  the  patient  suspected  to  be  suffering 
from  typhoid  fever  is  obtained  by  aseptic  puncture  of  the 
tip  of  a  finger,  or  of  a  vein  by  means  of  a  sterilized  syringe. 
The  blood  thus  obtained  is  permitted  to  flow  into  a  test- 
tube,  and  to  coagulate  in  a  slanting  position  ;  in  this  way 
the  largest  proportion  of  serum  will  be  obtained.  The  test  is 
then  made  according  to  one  of  the  methods  described  on 
page  64 ;  the  one  preferred  is  a  matter  of  indifference. 
The  desired  information  is  gained  most  quickly  through 
the  microscope,  and  only  by  this  means  is  it  possible  to 
determine  the  extreme  limit  of  agglutinating  activity — that 
is,  the  highest  degree  of  dilution  of  the  serum  with  which 
the  reaction  can  still  be  induced.  It  has  been  emphasized 
that  the  reaction  of  Gruber  is  a  quantitative  one,  and  that, 
therefore,  everything  depends  upon  determining  quantita- 
tively with  precision  the  agglutinating  activity  of  the 
serum.  A  proportion  of  i  to  50  is  sufficient  for  the  posi- 
tive diagnosis  of  typhoid  fever.  Many  thousand  specimens 
of  serum  have  now  been  examined  according  to  the  method 


TYPHOID   FEVER.  177 

of  Widal,  and  in  no  instance  has  this  dilution  yielded  the 
reaction  with  serum  not  from  a  case  of  typhoid  fever.  It  is 
possible,  however,  that  the  agglutinating  activity  in  cases 
of  typhoid  fever  may  be  below  these  figures  ;  with  dilu- 
tions between  i  :  50  and  i  :  10  Widal  and  Sicard  rec- 
ommend that  the  case  be  considered  as  suspicious  with 
regard  to  typhoid  fever,  and  that  the  observation  be  re- 
peated in  the  course  of  a  few  days.  The  serum  of  typhoid 
patients,  even  when  not  sterile,  retains  its  agglutinating 
power  unchanged  for  several  months.  It  may,  therefore,  be 
preserved  and  sent  from  one  place  to  another.  The  time 
when  the  agglutinating  property  appears  in  the  serum  is  of 
importance  in  diagnosis.  As  a  rule,  it  is  demonstrable 
after  the  seventh  day,  although  it  may  appear  later  or 
earlier.  The  earliest  that  it  has  been  observed  was  on  the 
second  day  by  C.  Frankel,  and  the  latest  in  the  first  days  of 
convalescence  by  Achard. 

The  Widal-Gruber  reaction  grows  feebler  in  the  first 
weeks  or  months  of  convalescence,  finally  to  disappear 
completely  in  some  cases.  Frequently,  however,  it  per- 
sists, and  it  may  be  demonstrable  after  the  lapse  of  years, 
or  even  of  decades,  and  it  can  thus  be  utilized  as  an  evi- 
dence that  the  individual  in  question  has  previously  suffered 
from  an  attack  of  typhoid  fever.  On  this  account  it  is 
absolutely  necessary,  in  the  application  of  serum-diagnosis, 
to  learn  from  the  patient's  history  whether  he  has  not 
already  at  some  time  passed  through  an  attack  of  typhoid 
fever,  however  mild  ;  otherwise,  there  is  danger  that  a  re- 
action of  older  date  may  be  employed  diagnostically  in 
relation  to  the  disease  under  observation.  Widal  and  Sicard 
divide  their  cases  of  typhoid  fever  into  five  groups,  accord- 
ingly as  the  serum  exhibits  a  greater  or  lesser  agglutinating 
activity.  In  the  first  group  the  agglutinating  power  is 
very  slight,  below  i  to  100;  in  the  second  group  it  is 
feeble,  between  i  to  100  and  i  to  200  ;  in  the  third  group 
it  is  moderately  great,  up  to  i  to  500  ;  in  the  fourth  group 
it  is  great,  up  to  i  to  2000  ;  and,  finally,  in  the  fifth  group 
it  is  above  i  to  2000.  Each  of  these  five  groups  includes 
both  mild  and  severe  cases.  The  agglutinating  curve  in 
individual  cases  of  typhoid  fever,  observed  throughout  the 
whole  course  of  the  disease,  likewise  exhibits  the  widest 
variations.  Each  case  bears  in  this  respect,  as  Widal  and 
Sicard  believe,   its   individual  impress.      Its    agglutinating 


178  CLINICAL  BACTERIOLOGY. 

power  may  appear  earlier  or  later,  and  in  greater  or  in  less 
degree,  and  it  may  even  be  entirely  absent,  as  these  observ- 
ers have  noted,  although  but  once  among  163  cases. 

The  examination  of  water  for  typhoid-bacilli  is  of 
great  practical  importance,  as  in  most  epidemics  the  drink- 
ing-water is  to  be  considered  as  the  vehicle  for  the  typhoid 
virus.  For  this  purpose  carbolic  acid  is  employed,  being 
added  to  the  suspected  water  in  such  amount  that  this  shall 
contain  from  0.05  to  0.25  per  cent,  of  the  acid.  This  addi- 
tion is  made  for  the  purpose  of  inhibiting  the  activity  of 
the  bacteria  present  in  water  that  liquefy  gelatin  ;  the 
typhoid-bacilli  themselves  readily  withstand  such  slight 
additions  of  carbolic  acid.  With  the  carbolized  water  three 
plates  are  made  in  the  customary  manner,  according  to  the 
method  of  Eisner.  As  by  this  means  only  small  amounts 
of  water  are  subjected  to  examination,  it  is  easily  possible 
that  typhoid-bacilli  may  escape  detection,  even  when  pres- 
ent. It  is,  therefore,  well  to  subject  considerable  amounts 
of  the  suspected  water  to  examination.  To  this  end  a 
sterilized,  alkaline,  concentrated  solution  of  peptone  and 
sodium  chlorid  is  employed  that  contains  in  a  specified 
number  of  cubic  centimeters  one  gram  of  peptone  and  one 
gram  of  sodium  chlorid.  This  amount  is  added  to  100 
cu.  cm.  of  the  carbolized  water  in  an  Erlenmeyer  flask, 
and  the  mixture  is  placed  in  the  thermostat  for  from  eigh- 
teen to  twenty-four  hours.  If  typhoid-bacilli  are  present 
in  the  water,  in  some  degree  protected  against  the  compe- 
tition of  the  other  bacteria  by  the  addition  of  carbolic  acid, 
they  undergo  multiplication,  and  they  can  be  more  readily 
demonstrated  on  plates  prepared  from  the  mixed  cultures. 
The  identification  with  certainty  of  the  developing  suspi- 
cious colonies  as  typhoid-colonies  is,  however,  again  at- 
tended with  considerable  difficulty.  The  water  contaminated 
by  typhoid  dejections  naturally  always  contains  also  the 
bacterium  coli  commune,  and  besides  there  are  frequently 
present  in  the  water  other  nonpathogenic  bacilli  that  bear 
an  extraordinary  resemblance  morphologically  and  in  cul- 
ture to  the  typhoid-bacillus — so-called  pseudotyphoid- 
bacilli.  These,  however,  sometimes  yield  the  indol-reaction 
and  sometimes  not.  A  considerable  number  of  these  bac- 
teria also  develop  in  the  mixed  cultures,  and  a  number 
even  much  better  than  the  specific  typhoid-bacilli.  A  de- 
cision as  to  the  presence   of  typhoid-bacilli  in  water  may 


TYPHOID   FEVER.  179 

therefore  be  finally  reached  only  when,  after  comparison  with 
an  unequivocal  pure  culture  (from  the  spleen  of  a  typhoid 
patient),  all  doubt  has  been  removed  that  both  the  culture 
obtained  from  the  water  and  the  earlier  pure  culture  agree 
in  every  detail,  and  with  regard  to  both  Pfeiffer's  and 
Gruber's  reactions.  Notwithstanding  these  difficulties, 
typhoid-bacilli  have  been  demonstrated  in  drinking-water 
in  several  instances  by  competent  observers. 

The  prophylaxis  of  typhoid  fever,  in  accordance  with 
what  has  already  been  said,  consists  especially  in  the  anti- 
sepsis of  the  sick-room.  The  feces  and  the  urine  from 
typhoid  patients,  all  materials  contaminated  by  these  dis- 
charges (body-clothing  and  bed-clothing,  etc.),  in  fact, 
everything  that  has  come  in  contact  with  the  patient,  must 
be  most  thoroughly  disinfected,  as  the  adherent  bacilli  may 
constitute  the  source  of  new  infections.  Methods  of  dis- 
infection are  described  in  the  Appendix. 

In  the  second  place,  the  prophylaxis  concerns  itself  prin- 
cipally with  the  hygienic  relations  of  the  drinking-water^ 
which  must  be  boiled  before  being  used  whenever  suspicion 
of  contamination  exists  in  times  of  epidemics. 

Immunity  and  Cure. — Typhoid  fever  is  one  of  those  dis- 
eases, as  shown  by  clinical  experience,  that  attack  the  same 
individual  but  once.  Two  attacks  have  occurred  in  the 
same  individual  in  about  two  per  cent,  of  all  the  cases  ; 
the  occurrence  of  three  attacks  in  the  same  individual  has, 
according  to  a  recent  report,  been  observed  only  five  times, 
and  four  attacks  in  the  same  individual  but  once.  It 
may,  therefore,  be  concluded  that  recovery  from  an  attack 
of  typhoid  fever  confers  a  certain  degree  of  immunity.  Sup- 
port for  this  view  is  found  in  the  fact  that  the  blood-serum  of 
some  individuals  who  have  recovered  from  typhoid  fever 
exhibits  immunizing  properties  with  relation  to  the  disease 
induced  experimentally  in  animals  with  typhoid-bacilli. 

The  immunization  of  animals  to  typhoid-bacilli  is  readily 
effected.  Bouillon-cultures  heated  to  a  temperature  of  60°  C. 
(140°  R),  or  agar-cultures  exposed  to  a  temperature  of  from 
54°  C.  (129.2°  F.)  to  56°  C.  (132.8°  F.)  have  been  employed 
for  this  purpose,  and  also  the  filtrate  of  unheated  virulent 
cultures,  or  of  cultures  in  thymus-bouillon.*     The  simplest 

■'^  According  to  a  recent  communication,  Buchner  and  Hahn  obtained  im- 
munity with  the  cell -juice  of  typhoid-bacteria  rubbed  up  and  expressed  by  the 
method  of  E.   Buchner  (p.  32).     (The   plasmatic  cell-juices  of  the  bacteria 


180  CLINICAL  BACTERIOLOGY. 

method  of  immunization  consists  in  the  employment  of  the 
ordinary  unheated  and  unfiltered  bouillon-culture.  Many 
experimental  animals  possess  a  considerable  degree  of 
immunity  to  the  typhoid-bacillus,  and  there  is  no  great  dif- 
ficulty in  increasing  this.  The  animal  is  treated  once  or 
twice  with  an  intraperitoneal  injection  of  half  that  amount 
of  bouillon-culture  that  is  just  necessary  to  cause  death. 
After  from  three  to  five  days  the  animal  will  be  able  to 
withstand  this  previously  lethal  amount,  and  after  several 
days,  one  and  a  half  times,  then  twice,  thrice,  etc.,  this 
dose  can  be  injected.  In  this  way  a  high  degree  of  immu- 
nity can  rapidly  be  induced.  The  blood-serum  of  the 
immunized  animals  is  in  turn  capable  of  conferring  immu- 
nity upon  untreated  animals. 

As  R,  Pfeififer  and  his  pupils  assume,  the  blood-serum  of 
typhoid  convalescents  and  of  animals  immune  to  typhoid 
fever  does  not  contain  antitoxic,  but  only  lysogenic  pro- 
tective substances  (p.  62) — that  is,  the  serum,  injected  into 
the  peritoneal  cavity  of  guinea-pigs  simultaneously  with 
living  typhoid-bacilli,  causes  dissolution  of  the  specific 
microorganisms.  If  two  milligrams  of  a  fresh,  virulent 
agar-culture  that  has  been  sterilized  by  exposure  for  several 
hours  in  the  thermostat  at  a  temperature  of  56°  C.  (132.8° 
F.)  are  injected  into  human  beings,  after  a  brief  period  of 
indisposition  the  blood-serum  of  such  persons  likewise 
possesses  lysogenic  properties.  The  strength  of  the  lyso- 
genicity  attains  the  same  degree  as  is  present  in  the  typhoid 
convalescent,  and  the  standard  of  the  serum  equals  about 
0.0 1  (p.  63).  If  the  appearance  of  the  specific  bactericidal 
(lysogenic)  substances  in  the  blood  of  individuals  that  have 
suffered  from  typhoid  fever  is  really,  as  Pfeiffer  assumes,  the 
essential  cause  of  immunity,  then,  according  to  the  experi- 
ments just  described,  it  must  also  be  possible,  by  means  of 
prophylactic  injections  of  minimal  amounts  of  the  dead 
bodies  of  typhoid-bacilli,  to  induce  immunity  of  like  degree 
and  duration.  The  decision  of  this  most  important  ques- 
tion must  await  the  results  of  further  investigation. 

Attempts  at  cure  with  protective  serum  or  with  milk  ob- 
tained from  an  immunized  animal  have  been  made  only  on  a 
small  scale,  and  distinct  success  has  not  as  yet  been  observed. 
This   is  in  harmony  with  the  view  alrea'dy  expressed   that 

are  designated  plasmins,  and  the  expressed  juice  of  typhoid-bacilli  correspond- 
ingly as  typhoplasmin.) 


ASIATIC   CHOLERA.  181 

the  serum  of  those  immune  to  typhoid  fever  does  not 
possess  antitoxic  properties.  The  therapeutic  experiments 
with  typhoid-cultures  exposed  to  a  temperature  of  60°  C. 
(140°  F.)  that  have  thus  far  been  made  upon  human  beings 
have  also  failed.  This  want  of  success  can  not  be  opposed 
to  the  possibility  already  suggested  of  prophylactic  immuni- 
zation by  means  of  dead  bacilli,  inasmuch  as  the  already 
diseased  and  poisoned  organism  may  react  differently  than 
the  healthy  body  to  injections  of  typhoid  poison. 


ASIATIC  CHOLERA. 

The  exciting  agent  of  Asiatic  cholera  is  the  comma- 
bacillus  discovered  by  Koch  in  1883. 

The  cholera-bacilli  are  more  or  less  markedly  curved  rods 
(vibrios) ,  from  one-half  to  at  most  two-thirds  as  large  as  tubercle- 
bacilli  (from  0.8  to  2  /a),  although  thicker  than  these.  The 
comma-form  is  not  well  defined  in  all  bacilli.  Every  preparation 
contains  forms,  especially  the  quite  young  bacilli,  that  appear  as 
simple,  straight,  extended  bacilli.  The  most  characteristic  and 
typical  commas  are  present  in  freshly  made  artificial  cultures. 
Besides,  the  form  of  the  growing  vibrios  varies  in  accordance  with 
their  source  in  one  or  another  epidemic.  In  certain  epidemics 
the  cholera-bacteria  assume  throughout  an  almost  straight  form. 
Frequently  the  comma-bacilli  are  arranged  in  pairs ;  when  two 
commas  are  so  applied  to  each  other  that  the  corresponding 
curves  are  opposed,  the  so-called  S-form  results. 

If  in  the  growth  of  the  vibrios  the  individual  newly  formed 
bacteria  adhere  to  one  another  after  division,  the  so-called 
cholera-spirilla  result.  Though  observed  very  seldom  in  the 
dejections  of  cholera-patients,  the  spirilla  occur  frequently  in 
artificial  cultures,  especially  when  these  have  become  old,  the 
nutrient  material  exhausted,  or  if  an  antiseptic  in  dilute  con- 
centration (as,  for  instance,  alcohol)  has  been  added.  The 
spirilla  occur  with  especial  frequency  in  the  peritonitic  exudate 
of  guinea-pigs  inoculated  with  cholera-bacilli.  The  spirilla  are 
generally  looked  upon  as  involution -forms,  principally  because 
they  are  thicker  in  cultures  than  the  young  individual  commas. 
The  cholera-vibrios  exhibit  extraordinarily  active  motility.  This 
is  especially  noticeable  in  the  hanging  drop,  in  which  their  ap- 
pearance suggests  a  swarm  of  dancing  gnats.  This  motility  is 
dependent  upon  the  presence  of  terminal  flagella  that  can  be 
readily  demonstrated  at  one  extremity  by  means  of  Loffler's 
method  of  staining. 


182  CLINICAL  BACTERIOLOGY. 

The  comma-bacillus  does  not  possess  spores.  The  arthro- 
spore-formation  that  Hiippe  assumed  to  exist  as  a  result  of 
his  earlier  investigations  has  not  been  confirmed  by  other  ob- 
servers. 

The  comma-bacilli  are  most  readily  stained  with  a  saturated 
aqueous  solution  of  fuchsin  or  with  carbolfuchsin.  The  expo- 
sure to  the  stain  should  be  longer  than  usual.  The  bacilli  do 
not  stain  by  Gram's  method. 

Cholera-bacteria  grow  upon  all  of  the  usual  nutrient  media, 
and  also  in  the  absence  of  oxygen  (facultative  anaerobiosis),  al- 
though, according  to  recent  investigations,  it  appears  that  oxygen 
can  never  be  entirely  wanting.  The  cholera-bacilli  always  require 
a  distinctly  alkaline  nutrient  medium  for  their  development,  as 
they  are  exceedingly  sensitive  to  the  presence  of  even  slight 


v.. 


^ 


>l 


-^S 


,  -i'-^d? 


Fig.  49.— Comma-bacilli  (from  the  mouth) ;  X  1000  (Giinther). 

amounts  of  acid.  The  degree  of  alkalinity  most  suitable  for  the 
cultivation  of  cholera-bacilli  is  secured  by  adding  one  gram  of 
crystallized  sodium  carbonate  to  loo  cu.  cm.  of  carefully  neu- 
tralized gelatin  (Dahmen),  or  by  preparing  a  10.6  per  cent, 
solution  of  soda  (from  calcined  sodium  carbonate)  and  adding 
55  cu.  cm.  of  this  to  one  liter  of  gelatin  (Fliigge).  For  ordi- 
nary purposes  simple  determination  of  the  alkalinity  by  means 
of  litmus-paper  is  sufficient,  but  the  paper  must  be  made  dis- 
tinctly blue. 

The  temperature-minimum  for  cholera-cultures  is  8°  C. 
(46,4°  F.)  ;  the  temperature-optimum  from  30°  C.  (86°  F. )  to 
40°  C.  (104°  F.). 

Appearances  of  Comma-bacilli  in  Cultures. —  Gelatin- 
plates. — The  plates  are  best  permitted  to  develop  at   22°  C. 


ASIATIC   CHOLERA.  183 

(71.6°  F.) — a  temperature  at  which  the  gelatin  still  retains  its 
solid  consistence.  After  from  twenty-four  to  thirty  hours  the 
colonies  appear,  on  microscopic  examination,  as  small,  whitish- 
yellow  dots,  with  an  irregular,  rough  margin.  Their  contents 
are  coarsely  granular.  After  a  time  the  granules  become  glis- 
tening, so  that  the  colonies  appear  as  if  they  had  been  strewn 
with  small  bits  of  glass.  With  the  further  development  of  the  cul- 
ture the  gelatin  undergoes  liquefaction,  and  this  occurs  the  more 
rapidly  the  more  favorable  the  temperature  and  the  more  nearly 
the  degree  of  alkalinity  approximates  its  optimum.  At  the  be- 
ginning liquefaction  progresses  quite  slowly;  small,  funnel-shaped 
depressions  constitute  the  first  peculiarity  visible  to  the  naked  eye. 
Viewed  with  oblique  light,  the  plate  appears  as  if  it  has  been 
punctured  superficially  with  a  fine  needle.  This  beginning  lique- 
faction is  characterized  by  a  bright  boundary  surrounding  the  in- 
dividual colonies,  as  viewed  with  low  powers  of  the  microscope. 
The  colonies  have  now  become  somewhat  darker  and  opaque, 
and  their  irregular  margin  is  not  rarely  marked  by  fine,  pointed 
processes.  Later  the  liquefaction  becomes  more  active,  the 
funnel-shaped  depressions  become  larger,  and  the  colonies  sink 
to  the  bottom  of  the  depressions.  The  border  surrounding  the 
colony  (area  of  liquefaction)  is  no  longer  bright,  but  filled  with 
small,  grayish  masses.  These  consist  of  groups  of  bacteria  that 
have  become  detached  from  the  colony  and  admixed  with 
the  liquefied  gelatin.  The  colony  itself,  a  brown,  irregular 
mass,  lies  at  the  bottom  of  the  funnel.  In  order  to  bring  it 
clearly  into  view,  the  tube  of  the  microscope  must  be  pushed 
downward.  In  employing  gelatin  prepared  according  to  the 
method  of  Forster  (p.  81)  the  plates  may  be  exposed  to  a  tem- 
perature of  25°  C.  (77°  F.)  or  26°  C.  (78.8°  F.).  Under 
these  circumstances  growth  takes  place  much  more  actively, 
and  the  peculiarities  of  the  colonies  described  appear  much  more 
quickly. 

Gelatin  Stab-culture. — Growth  takes  place  along  the  entire 
line  of  inoculation  in  the  form  of  a  white  thread  that  grows 
thinner  downward.  After  from  twenty-four  to  forty-eight  hours 
liquefaction  slowly  sets  in  in  the  upper  portions,  and  here  also 
leads  to  the  formation  of  a  funnel.  This  is  naturally  more  ex- 
tensive than  the  funnel  of  liquefaction  of  the  individual  colonies 
in  plates.  Liquefaction  takes  place  so  slowly  that  at  first  the 
fluid  formed  has  time  to  undergo  evaporation.  The  upper  por- 
tion of  the  liquefaction-funnel  is,  therefore,  empty,  and  an  ap- 
pearance is  created  as  if  the  puncture  of  inoculation  contained 
an  air-bubble.  The  line  of  inoculation  itself  appears  in  slight 
degree  liquefied,  and,  in  consequence,  somewhat  enlarged.  Its 
lower  portion  contains  the  bacterial  masses  that  have  gravi- 
tated thither,   and  which  here  assume  the  form  of  a  spirally 


184  CLINICAL  BACTERIOLOGY. 

wound  thread.  In  the  further  course  of  the  growth — not  before 
several  weeks — the  gelatin  is  completely  liquefied. 

On  agar-plates  growth  is  not  so  characteristic  as  in  gelatin- 
plates.  The  superficial  colonies  present  a  peculiar,  light  gray- 
ish-brown, transparent  appearance. 

Agar  streak- cultures  exhibit  a  grayish-white,  moist,  glistening 
coating. 

Blood- serum  is  gradually  liquefied. 

Potatoes. — In  spite  of  the  usually  acid  reaction  of  potatoes 
the  cholera-vibrios  generally  thrive  upon  this  culture-medium, 
but  only  at  temperatures  above  21°  C.  (69.8°  F.).  They  then 
form  a  grayish  or  grayish-brown,  thin,  translucent  coating. 
Upon  some  varieties  of  potatoes,  however,  the  comma-bacilli  do 
not  thrive,  but  they  can  readily  be  cultivated  if  the  potatoes  are 
rendered  slightly  alkaline  by  means  of  a  solution  of  soda,  or  if 
they  are  boiled  in  a  three  per  cent,  solution  of  sodium  chlorid. 

Milk  is  coagulated  by  cholera-bacteria  obtained  in  certain 
epidemics,  but  not  by  others.     The  latter  appears  to  be  the  rule. 

Bouillon  is  rendered  turbid,  and  in  the  majority  of  cases  a 
superficial  membrane  forms  at  body-heat.  Like  the  majority 
of  vibrios  and  spirilla,  the  cholera-vibrios  also  possess  the  prop- 
erty of  multiplying  with  especial  activity  in  quite  dilute  bouillon 
(from  six  to  eight  times).  A  one  per  cent,  aqueous  solution  of 
peptone,  with  addition  of  one-half  per  cent,  sodium  chlorid, 
also  favors  the  development  of  the  comma-bacilli.  If  the  pep- 
tone is  not  alkaline  originally,  the  last-mentioned  nutrient 
medium  must  be  rendered  so  by  addition  of  soda. 

Cholera-red  Reaction. — If  a  few  drops  of  pure  dilute  hydro- 
chloric acid  or  sulphuric  acid  are  added  to  cholera-cultures  that 
have  grown  in  nutritive  media  containing  peptone,  a  rose-red  or 
purple-red  coloration  appears  within  a  short  time.  Bouillon- 
cultures  yield  this  reaction  after  exposure  for  twelve  hours  in 
the  thermostat.  This  so-called  cholera-red  reaction  is  nothing 
more  than  an  ordinary  nitroso-indol  reaction.  The  comma- 
bacilli  possess  the  property  of  forming  indol  and  of  converting 
into  nitrites  the  nitrates  always  present,  at  least  in  small  amount, 
in  nutrient  solutions.  In  addition  to  the  comma-bacilli,  there 
are  other  vibrios  that  likewise  yield  the  nitroso-indol  reaction  : 
as,  for  instance,  the  vibrio  Metschnikoff,  and  the  vibrio  bero- 
linensis  found  in  the  water-supply  of  Berlin.  (See  water-bac- 
teria. Appendix.)  The  Finkler-Prior  bacillus  and  the  Denecke 
cheese-bacillus  also  form  indol,  but  no  nitrite,  so  that  addition 
of  pure  acids  free  from  nitrous  acid  does  not  cause  a  red  colora- 
tion with  them.  The  one  per  cent,  aqueous  solution  of  peptone 
and  sodium  chlorid  previously  mentioned  is  especially  suited 
for  the  formation  of  cholera-red.  The  reaction  is  at  times  want- 
ing in  bouillon  when  too  much  or  too  little  nitrate  is  present. 


ASIATIC   CHOLERA.  185 

Tenacity  of  the  Cholera- vibrios. — The  comma-bacilli 
are  endowed  with  extremely  little  resistance.  They  can  be 
destroyed  in  four  minutes  in  ivater  at  a  temperature  of  52° 
C.  (125.6°  F.).  Lower  temperatures  are,  however,  better 
borne,  although  the  bacilli  die  in  ice  after  the  lapse  of  a 
few  days.  The  susceptibility  of  comma-bacilli  to  small 
amounts  of  acids  has  already  been  mentioned.  The  ad- 
dition of  not  more  than  0.07  or  0.08  per  cent,  of  hydro- 
chloric or  nitric  acid  to  neutral  culture-media  is  capable  of 
preventing  all  development.  These  facts  explain  why  the 
normal  gastric  juice,  with  its  hydrochloric-acid  content  of 
about  0.2  per  cent.,  constitutes  an  insurmountable  obstacle 
to  the  cholera-vibrios.  •  If  cholera-bacilli  are  spread  in  a 
thin  layer  upon  a  basis  of  any  sort  so  that  they  are  ren- 
dered completely  adherent  and  dry,  they  lose  their  capa- 
bility of  development  in  the  course  of  three  hours.  Dried 
upon  the  hand,  they  survive  only  for  one  or  two  hours. 
The  same  rapid  destruction  (in  any  event  within  twenty -four 
hours)  takes  place  in  connection  with  the  contamination  of 
smooth  swfaces — such  as  floors,  paper,  etc. — with  comma- 
bacilli.  From  these  considerations  it  may  be  concluded 
that  the  transmission  of  cholera  by  way  of  the  air,  through 
the  intermediation  of  dried  particles  of  dust,  is  scarcely 
possible.  Surrounded  by  moisture  the  comma-bacilli  may, 
under  favorable  conditions,  retain  their  vitality  for  a  long 
time,  up  to  nine  months — as,  for  instance,  in  moist  linen 
rolled  together  compactly  and  kept  in  a  cool  place,  and 
containing  the  vibrios  in  pure  culture,  so  that  they  are  not 
overgrown  by  other  bacteria.  They  may  be  found  alive  in 
agar-cultures  and  gelatin-cultures  after  the  lapse  of  six 
months.  The  usual  antiseptics,  even  in  feeble  concentration, 
destroy  the  cholera-vibrios  within  a  short  time  :  one-half  per 
cent,  carbolic  acid,  for  instance,  in  the  course  of  a  few 
minutes.  In  fresh  milk  the  comma-bacilli  retain  their 
vitality  for  twenty-four  hours  ;  in  boiled  milk,  for  two  or 
three  days  ;  upon  articles  of  food  protected  from  evapora- 
tion under  a  glass  jar,  for  from  four  to  eight  days.  The 
bacilli  sometimes  survive  for  weeks  in  the  dejections  of 
cholera-patients,  although  this  occurs  only  under  peculiarly 
favorable  conditions.  In  sterilized  water,  from  whatever 
source,  living  cholera-vibrios  can  be  demonstrated  after  the 
lapse  of  months.  In  unsterilized  water,  on  the  other  hand, 
and  especially  in  bacterial  mixtures,  in  which  the  comma- 


186  CLINICAL  BACTERIOLOGY. 

bacilli  enter  into  contest  with  other  microbes,  the  conditions 
vary  in  accordance  with  the  external  temperature  and  the 
amount  of  sodium  chlorid  present  in  the  fluid  in  question. 
The  high  temperature  of  summer  and  increase  in  the  amount 
of  sodium  chlorid  favor  the  development  of  cholera-bacilli, 
while  at  low  atmospheric  temperatures,  or  in  the  presence 
of  a  small  amount  of  sodium  chlorid,  they  are  quickly  over- 
grown by  the  associated  microorganisms.  If  cholera- 
dejections  gain  entrance  into  streams,  the  bacilli  mostly 
adhere  to  the  particles  of  mucus,  which  they  utilize  as  a 
nutrient  medium.  They  are  thus  often  removed  from  the 
influence  of  the  current  and  the  competition  of  other  bac- 
teria, and  they  not  rarely  maintain  their  vitality  in  streams 
for  a  long  time  in  spite  of  the  unfavorable  temperature  and 
the  process  of  self-purification. 

The  Occurrence  of  Cholera- vibrios. — Comma-bacilli 
are  found  constantly  in  all  cases  of  Asiatic  cholera,  and 
in  innumerable  amount ;  in  part,  in  pure  culture  in  the 
liquid  intestinal  contents  ;  further,  in  the  intestinal  walls  of 
patients';  and  only  exceptionally  in  other  organs.  The 
vibrios  can  be  demonstrated  in  the  feces  on  an  average  to 
the  tenth  day  after  the  inception  of  the  disease ;  not 
rarely,  however,  for  a  longer  time,  and  sometimes  after  the 
termination  of  the  disease  (from  the  forty-sixth  to  the  forty- 
eighth  day  of  convalescence).  Comma-bacilli  have  never 
been  demonstrated  in  association  with  other  diseases.  Dur- 
ing recent  epidemics  true  cholera-bacilli  were  found  repeat- 
edly in  the  diarrheal  stools  of  patients  whose  disease  clini- 
cally pursued  a  mild  course  and  apparently  bore  no  relation 
to  Asiatic  cholera.  It  is  possible,  however,  that  these  cases 
represented  the  mildest  grades  of  cholera,  caused  by  bac- 
teria of  low  degrees  of  virulence.  Further,  comma-bacilli 
were  found  in  some  cases  in  the  solid  stools  of  healthy  per- 
sons subjected  to  bacteriologic  examination  only  because 
their  relations  brought  them  into  the  environment  of 
cholera-patients,  or  because  they  had  drunk  suspected 
water,  but  who  presented  no  symptoms  of  disease  whatever. 
It  is  probable  that  in  these  cases  there  existed  a  natural 
immunity  that  permitted  the  passage  of  the  vibrios  through 
the  intestinal  canal  without  injury. 

Outside  the  human  body  the  cholera-bacillus  of  Koch 
has  been  found  in  stagnant  water  in  India,  where  cholera  is 
epidemic.      In   recent    epidemics   of  cholera  comma-bacilli 


ASIATIC  CHOLERA.  187 

were  repeatedly  demonstrated  in  the  water-supply  through 
conduits,  in  the  water  of  improperly  situated  irrigation-fields 
(Nietleben),  in  the  water  of  streams,  and  in  the  bilge-water  of 
ships.  The  waters  in  question  all  bore  some  close  relation  to 
cholera-foci,  having  been  contaminated  by  the  dejections  of 
the  first  cases  of  the  disease  introduced,  so  that  in  this  way 
they  were  capable  of  constituting  the  source  for  the  further 
spread  of  the  disease.  These  facts  demonstrate  that  the 
cholera-bacillus  may  pass  a  saprophytic  existence.  Forms 
of  comma-bacilli  have,  further,  been  cultivated  from  the 
Spree,  the  Elbe,  the  Danube,  and  the  Seine,  that,  in  their 
microscopic  and  cultural  appearances,  closely  agreed  with 
the  vibrio  of  Koch,  but  whose  relation  to  existing  cases  of 
cholera  was  not  so  easily  demonstrable.  These  water- 
vibrios  are  in  part  to  be  distinguished  from  the  bacillus  of 
Koch  by  the  absence  of  pathogenicity ;  others,  however, 
are  toxic  for  animals.  Thus,  virulent  vibrios  were  found 
almost  regularly  in  the  contents  of  Paris  sewers  in  the 
summer  of  1892,  although  at  the  time  there  was  not  a  sin- 
gle case  of  cholera  in  Paris.  All  of  these  bacteria,  however, 
exhibit  differences  as  compared  with  the  vibrio  of  Koch, 
even  though  they  are  often  slight  and  difficult  of  dem- 
onstration. A  bacterium  absolutely  identical  with  the 
cholera-bacillus,  but  in  its  source  without  any  relation  to 
cholera,  has  never  been  found. 

There  are  two  methods  of  making  with  certainty  the 
differential  diagnosis  between  the  true  exciting  agent  of 
cholera  and  the  vibrios  resembling  it.  These  are  the  reac- 
tions of  Pfeiffer  and  Gruber  (pp.  62  and  63).  In  the  per- 
formance of  Pfeiffer's  test  the  blood-serum  of  guinea-pigs 
or  of  other  animals  that  have  been  highly  immunized  to 
cholera  is  diluted  with  ordinary  bouillon  in  the  proportion  of 
I  :  TOO.  To  one  cubic  centimeter  of  this  mixture  are  added 
about  two  milligrams  of  the  vibrios  to  be  examined,  caught 
up  with  a  platinum  loop,  and  the  whole  is  injected  into  the 
abdominal  cavity  of  a  young  guinea-pig  weighing  about 
200  grams.  By  means  of  delicate  capillary  glass  tubes 
specimens  of  the  peritoneal  exudate  that  at  once  forms  are 
removed  at  intervals  of  five  minutes  and  examined,  both 
stained  and  unstained.  If  the  suspected  organisms  are  true 
cholera-vibrios,  the  bacilli  are  shortly  seen  to  become 
immobile,  later  collected  in  small  spherules,  and  finally, 
within  twenty  minutes,  completely  dissolved.     If,  however, 


188  CLINICAL   BACTERIOLOGY. 

this  phenomenon  does  not  take  place,  the  vibrio  in  question 
is  not  the  cholera-organism.  One  source  of  error  must, 
however,  always  be  borne  in  mind,  if  Pfeiffer's  reaction 
proves  positive  :  It  is  possible  that  the  organisms  present  are 
attenuated,  to  a  certain  degree  saprophytic,  vibrios,  that 
are  dissolved  in  consequence  of  the  normal  bactericidal 
activity  of  the  organism  of  the  guinea-pig,  in  the  absence 
of  any  specific  serum.  To  avoid  this  error,  a  platinum 
loopful  of  the  culture  in  question  added  to  one  cubic  centi- 
meter of  normal  serum-bouillon  mixture  (i  :  lOo)  is  in- 
jected into  the  peritoneal  cavity  of  a  control  guinea-pig.  If, 
after  the  lapse  of  twenty  minutes,  the  vibrios  are  found 
living  and  motile  in  the  control-animal,  whereas  those 
treated  with  cholera-serum  are  destroyed,  then  the  diagnosis 
of  Asiatic  cholera  may  be  made  with  certainty. 

The  second  test,  that  of  Gruber,  is  more  readily  per- 
formed, as  experiment  on  an  animal  is  not  required.  The 
suspected  vibrios*  may  be  added  to  the  serum  of  an 
animal  rendered  immune  to  cholera  in  the  proportions 
of  I  to  50,  I  to  100,  and  above  ;  and  the  mixture  is  studied 
at  once  with  high  powers  of  the  microscope.  If  the 
vibrios  lose  their  motility ;  if  they  collect  in  groups  ; 
if,  thus,  agglutination  takes  place — then  the  suspected 
microorganisms  are  true  cholera-vibrios.  One  of  the 
macroscopic  agglutination-tests  (p.  64)  may  be  made — e.g., 
bouillon  is  inoculated  with  the  suspected  vibrios  and  the 
serum  of  an  animal  immune  to  cholera  is  added  in  the  pro- 
portion named.  If,  after  the  lapse  of  from  sixteen  to 
twenty-four  hours,  the  vibrios  have  formed  a  flocculent  pre- 
cipitate at  the  bottom  of  the  test-tube,  while  the  overlying 
fluid  has  become  clear,  then  again  the  suspected  organisms 
are  true  cholera-bacilli.  Gruber's  reaction  has  the  advan- 
tage of  being  independent  of  the  virulence  of  the  micro- 
organisms, and,  besides,  it  renders  unnecessary  the  induc- 
tion of  a  high  degree  of  immunity  in  the  animals  yielding 
the  blood-serum. 

The  Development  of  Cholera. — Infection  takes  place 
invariably  by  way  of  the  mouth,  the  bacilli  being  taken  up 
with  articles  of  food,  and  principally  with  drinking-water. 
Infection  through  the  air  is  possible  only  in  the  immediate 
neighborhood  of  the  source  of  infection.  The  rapid  death 
of  the  germs  on  drying  renders  improbable  the  transmission 
of  the  disease   for  long   distances  by  means  of  dust,  etc. 


ASIATIC  CHOLERA.  189 

The  inhaled  bacilli  must  also  be  restrained  in  the  mouth,  as 
infection  does  not  take  place  through  the  lungs.  From  the 
mouth  the  vibrios  gain  entrance  into  the  stomach.  If  the 
hydrochloric-acid  content  of  the  stomach  is  normal,  the 
vibrios  succumb  to  it.  If  infection  takes  place,  it  may  be 
assumed  that  either  large  amounts  of  the  infected  water 
have  been  drunk,  so  that  some  of  the  bacteria  escape 
the  action  of  the  hydrochloric  acid,  in  consequence  of  the 
marked  dilution  of  the  gastric  contents,  or  that  the  function 
of  the  stomach  was  impaired  from  some  cause,  and  the 
hydrochloric-acid  content  was  subnormal.  Having  gained 
entrance  into  the  intestine,  the  bacteria  multiply  and  give 
rise  to  the  production  of  toxins.  Mere  multiplication  of 
the  bacteria  in  the  intestine  does  not  constitute  cholera. 
Such  an  effect  was  observed  in  the  well-known  experiments 
of  Pettenkofer,  without  the  development  of  actual  cholera. 
Only  when  sufficient  toxin  has  been  produced  to  cause 
injury  of  4Jie  intestinal  mucous  membrane  and  when  the 
toxi7t  is  absorbed  does  the  disease  develop.  While  the  tis- 
sues and  the  blood  become  impoverished  in  water,  the  pro- 
fuse rice-water  stools  take  place,  and  with  them  innumer- 
able bacilli  are  evacuated. 

The  toxic  action  is  manifested  especially  in  the  constitu- 
tional symptoms  (feebleness  of  heart,  decline  of  temperature, 
etc.).  Cholera-typhoid  also  is  now  generally  looked  upon 
as  an  intoxication,  and  the  renal  disease  complicating 
cholera  likewise  depends  in  part  upon  the  toxic  activity  of 
the  comma-bacilli,  being  caused  in  part  by  the  ischemia  re- 
sulting in  consequence  of  the  withdrawal  of  water. 

Brieger  and  Frankel  have  demonstrated  the  presence  of 
a  toxalbumin  in  cholera-cultures,  but  greater  significance  in 
experiments  on  animals  has  been  attached  to  the  poison 
contained  within  the  bodies  of  the  bacilli  themselves,  and 
whose  effects  have  been  studied,  especially  by  R.  Pfeiffer. 
If  a  young  agar  streak-culture,  from  twenty  to  twenty-four 
hours  old,  is  destroyed  by  exposure  for  ten  minutes  to  the 
action  of  chloroform,  and  ten  milligrams  of  the  bodies  of 
the  vibrios  thus  destroyed  are  injected  into  the  peritoneal 
cavity  of  a  guinea-pig,  the  animal  will  die.  After  the  lapse 
of  two  hours  it  becomes  relaxed,  its  temperature  falls  below 
30°  C.  (86°  F.),  and  death  takes  place  in  the  course  of 
eight  or  ten  hours,  usually  amid  violent  clonic  convul- 
sions.    The  poison  that  thus  adheres  to  the  bodies  of  the 


190  CLINICAL  BACTERIOLOGY. 

bacteria  is  of  most  evanescent  nature.*  It  does  not  with- 
stand exposure  to  temperatures  above  60°  C.  (140°  F.), 
drying  and  the  Hke.  If  the  cultures  are  boiled  for  several 
hours,  according  to  the  view  of  R.  Pfeiffer  secondary  toxic 
substances  are  formed  that  manifest  their  activity  only  in 
much  larger  amounts.  The  intoxication,  however,  pre- 
sents the  clinical  features  described.  This  intoxication  with 
the  intracellular  cholera-poison  is  remarkable  for  the  rapidity 
with  which  it  appears,  and  for  the  entire  absence  of  any 
period  of  incubation,  such  as  is  commonly  observed  in  con- 
nection with  the  diphtheria-toxin  and  the  tetanus-toxin.  The 
severity  of  the  intoxication  depends  upon  the  amount  of  toxin 
and  the  rapidity  of  absorption.  The  effects  are  most  quickly 
manifested  when  the  poison  is  introduced  directly  into  the 
blood-stream. 

Epidemics  of  cholera  arise,  according  to  Koch,  from 
the  first  imported  case,  through  whose  dejections,  con- 
taining the  bacilli,  the  disease  is  spread.  The  type  of 
distribution  may  be  of  two  kinds.  The  disease  may  spread 
in  foci  :  a  member  in  the  family  of  the  patient  first  attacked 
is  seized,  then  others,  then  another  family  in  the  same  house, 
a  neighbor,  a  stranger  accidentally  present  in  the  house, 
the  laundress  to  whom  the  linen  from  the  first  case  is  sent ; 
in  this  way  each  disease-focus  gives  rise  to  another,  and  all 
together  form  a  closed  chain.  In  every  instance  infection 
has  taken  place  through  contamination  with  the  dejections 
of  an  earlier  case.  Often  enough  the  connection  of  the 
individual  cases  with  one  another  is  not  demonstrable. 
Thus,  for  instance,  insects  may  carry  the  disease-germs 
great  distances  and  deposit  them  upon  articles  of  food  that 
seemingly  have  in  no  way  come  in  contact  with  a  focus 
of  disease ;  or  apparently  healthy  individuals  from  the 
neighborhood  of  the  person  first  attacked  may  disseminate 
the  germs  through  their  infected  dejecta. 

On  the  other  hand,  the  outbreak  of  the  disease  may  take 
place  in  an  explosive  manner,  large  numbers  of  cases  appear- 
ing equably  and  simultaneously  throughout  a  town  or  a 
city.  This  occurs  when  the  water,  whether  delivered 
through  conduits  or  obtained  from  streams,  is  infected  by 
cholera-dejections,  and  the  germ  is  thus  capable  of  being 
equably  spread  over  the  entire  city,  and  carried  into  every 

*  The  poison  can  also  be  expressed  from  the  bodies  of  the  bacteria  accord- 
ing to  the  method  of  E.  Buchner,     (See  foot-note,  p.  179.) 


ASIATIC   CHOLERA.  191 

household.  The  cholera-bacillus  has  never  been  demon- 
strated in  the  air  or  in  the  soil,  which  likewise  might  be 
considered  as  sources  of  such  explosive  outbreaks  of  cholera. 
The  bacillus  has  been  found  during  recent  epidemics  in 
river-water  and  in  conduits. 

Epidemics  of  cholera  do  not  always  conform  strictly  to 
one  or  the  other  of  the  two  types  described  by  Koch  ;  one 
may  be  combined  with,  or  pass  over  into,  the  other,  etc. 
Individual  predisposition,  the  quantity  and  quality  of  food, 
the  density  of  population,  individual  and  civic  cleanliness, 
distinctly  influence  the  distribution  of  the  disease. 

The  foregoing  theories,  representing  essentially  Koch's 
views,  are,  in  fact,  capable  of  explaining  almost  all  of  the 
manifestations  of  recent  cholera-epidemics.  One  point  only, 
however,  is  obscure  :  namely,  the  question  why  in  some  in- 
stances an  epidemic  does  not  occur.  Thus,  Paris  escaped 
in  1892,  although  the  bacilli  were  present  in  the  sewers; 
and  only  a  few  cases  occurred  in  Berlin  in  the  year  1893, 
although  the  waterways  were  known  to  be  infected.  It  is 
possible  that  the  energetic  intervention  on  the  part  of  the 
authorities,  the  filtration  of  the  water,  as  well  as  the  careful 
observance  of  all  hygienic  regulations,  prevented  the  irrup- 
tion of  an  epidemic.  The  thought,  however,  can  not  be  en- 
tirely put  aside  that  possibly  the  *'  local  predisposition  " 
was  wanting.  According  to  Pettenkofer,  for  the  occurrence 
of  an  epidemic  a  local  and  a  temporal  predisposition  are 
necessary.  *'  The  cholera-germ  (x)  forms  upon  the  basis 
of  the  local  and  the  temporal  predisposition  of  the  soil  (j) 
the  cholera-poison  (^)."  According  to  this  view,  the  dis- 
ease can  never  be  transmitted  from  one  human  being  to  an- 
other, but  the  germ  must  first  mature  in  the  earth,  and  then 
the  poison  is  taken  up  by  the  lungs.  In  view  of  the  facts 
mentioned  this  theory  of  Pettenkofer's  can  scarcely  be  main- 
tained any  longer  as  against  the  bacillus  of  Koch.  A 
number  of  epidemiologic  facts,  however,  indicate  that,  in 
addition  to  the  comma-bacillus,  some  other  not  yet  suffi- 
ciently determined  influences '  are  necessary  for  the  occur- 
rence of  an  epidemic. 

Experiments  upon  Animals  and  Human  Beings. — Even 
though  some  uncertainty  exists  with  regard  to  the  last 
point,  there  can,  however,  be  no  further  doubt  with  regard 
to  the  etiologic  significance  of  the  comma-bacillus  as  the 
exciting  agent  of  cholera.     The  final  evidence  for  this  was 


192  CLINICAL  BACTERIOLOGY. 

furnished  by  the  experiments  upon  human  beings  that  were 
undertaken  in  part  unintentionally  (accidental  laboratory- 
infection),  in  part  intentionally.  The  introduction  of  the 
bacilli  into  the  stomach  may  be  unattended  with  any  effect ; 
at  times  it  gives  rise  to  more  or  less  intense  diarrhea  (auto- 
infection  of  Pettenkofer  and  Emmerich)  ;  in  other  cases — 
as,  for  instance,  in  one  reported  by  Metschnikoff — however, 
it  induces  true  dangerous  cholera,  with  all  its  clinical  symp- 
toms. The  sad  fate  of  a  young  Hamburg  physician  is  well 
known,  who  died  of  typical  cholera  after  a  drop  of  peri- 
toneal exudate  containing  vibrios  had  entered  his  mouth 
in  the  performance  of  Pfeiffer's  test.  Subcutaneous  inocu- 
lation with  cholera-bacilli  causes  in  human  beings  only 
moderate  local  symptoms  and  fever  of  brief  duration.  The 
blood  under  these  circumstances  acquires  immunizing  prop- 
erties (G.  Klemperer). 

A  disease  resembling  cholera  can  be  induced  in  guinea- 
pigs  by  direct  introduction  of  the  vibrios  into  the  duodenum, 
by  avoidance  of  the  stomach,  after  ligation  of  the  choledoch 
duct ;  or  by  introduction  of  the  bacilli  into  the  stomach 
after  previous  alkalinization  of  the  gastric  contents  by  means 
of  soda-solution  and  injection  of  two  or  three  cubic  centi- 
meters of  tincture  of  opium  into  the  peritoneal  cavity.  The 
object  of  ligating  the  choledoch  duct,  as  well  as  of  the  in- 
jection of  the  opium,  is  to  inhibit  the  peristaltic  activity  of 
the  intestine.  Similar  morbid  manifestations — cholera-like 
stools,  decline  of  temperature,  etc. — can  be  induced  in 
guinea-pigs  and  rabbits  by  intravenous  introduction  of  the 
comma-bacilli,  especially  after  previoiis  intoxication  with 
alcohol,  or  by  simultaneous  injection  of  comma-bacilli  and 
metabolic  products  of  varieties  of  proteus. 

Intraperitoneal  injection  of  virulent  cholera-vibrios  is  fol- 
lowed in  guinea-pigs  (a  platinum  loopful  containing  about 
2.5  mg.  of  bacterial  colonies  scraped  from  the  surface  of 
agar-agar  for  a  guinea-pig  weighing  from  300  to  500  grams) 
by  death,  preceded  by  paralytic  phenomena  and  rapid  de- 
cline of  temperature.  There  takes  place  under  these  con- 
ditions, as  has  already  been  stated,  an  intoxication  with  the 
poisons  contained  within  the  bodies  of  the  comma-bacillus 
(p.  189).  In  the  digestive  tract  of  guinea-pigs  destroyed 
by  this  means  special  changes  are  usually  not  to  be 
observed.  At  times,  the  large  bowel  is  markedly  injected 
and  the  small  intestine  is  not  rarely  filled  with  grayish  fluid 


ASIATIC  CHOLERA.  193 

containing  large  numbers  of  comma-bacilli.  On  the  whole, 
however,  it  is  scarcely  possible  to  insist  upon  a  complete 
analogy  between  the  intoxication  of  guinea-pigs  and  cholera 
as  it  appears  in  human  beings. 

Bacteriologic  Diagnosis  of  Cholera. — If  an  attack  of 
disease  characterized  by  the  occurrence  of  profuse,  rice- 
water  diarrhea,  vomiting,  decline  of  temperature,  cramps  in 
the  calves,  etc.,  arouse  suspicion  of  cholera — a  suspicion 
that  must  be  considered  and  investigated  in  every  instance, 
especially  in  travelers  during  the  summer  season,  and  par- 
ticularly when  cholera  exists — bacteriologic  examination 
of  the  stools  is  an  absolute  duty.  The  procedure  in  a 
case  suspected  to  be  one  of  cholera  is  as  follows  : 

1.  Microscopic  Examination. — Cover-slip  preparations  are 
made  with  a  flake  of  mucus  from  the  feces,  and  stained 
with  dilute  carbol-fuchsin  solution.  The  diae^nosis  of 
Asiatic  cholera  is  rendered  in  the  highest  degree  probable 
if  the  vibrios  form  masses  "  in  which  the  individual  bacilli 
all  point  in  the  same  direction,  so  that  an  appearance  is 
created  as  if  a  small  swarm  of  them,  somewhat  like  fish  in 
a  slowly  flowing  stream,  are  following  one  another";  or 
if,  "  in  addition  to  isolated  bacteria  presenting  the  appear- 
ance of  cholera-bacteria,  only  the  bacterium  coli  is  found.  " 
The  probable  diagnosis  of  cholera  can  be  made  microscop- 
ically in  about  50  per  cent,  of  suspected  cases,  but  in  every 
instance  it  should  be  confirmed  by  cultural  investigation. 

2.  Cidtitral  Investigation. — {a)  Gelatin-plate  Method. — 
Three  gelatin-inoculations  are  prepared,  in  the  usual  man- 
ner, from  the  feces,  if  possible  from  a  flake  of  mucus,  and 
the  inoculated  culture-media  are  poured  into  three  Petri 
dishes  ;  these  are  kept  at  a  temperature  of  from  22°  C. 
(71.6°  F.)  to  26°  C.  (78.8°  F.).  If  upon  these  plates 
colonies  are  found  after  from  fourteen  to  thirty  or  forty- 
eight  hours,  with  uneven,  rough  borders,  strewn  with  bright 
granules  resembling  fragments  of  glass,  and  presenting  an 
area  of  liquefaction,  and  if  these  colonies  prove  to  be 
constituted  of  commas,  a  high  degree  of  probability  is 
given  the  diagnosis,  and  it  is  scarcely  possible  that  the  dis- 
ease under  consideration  is  any  other  than  Asiatic  cholera. 
So  far  as  is  yet  known,  there  occur  in  the  human  intestine 
no  vibrios  other  than  the  comma-bacilli  that  ^w^  rise  to 
colonies  presenting  the  characteristics  that  have  been  de- 
scribed. 

13 


194  CLINICAL  BACTERIOLOGY. 

(J?)  Peptone -culture  (Fertilizing  Method,  SchotteliuSy 
Koch). — The  plate-method,  which  yields  admirable  results 
in  the  diagnosis  of  marked  cases  of  Asiatic  cholera,  does 
not  suffice,  however,  for  other  cases  in  which  the  dejections 
contain  only  a  small  number  of  comma-bacilli.  This  small 
number  of  vibrios  are  overrun  on  plates  by  the  fecal  bac- 
teria, and  do  not  develop  at  all.  It  is,  therefore,  necessary 
in  every  suspected  case  to  adopt,  in  addition  to  the  original 
plate-method,  a  further  method  of  investigation  :  namely, 
the  preparation  of  peptone-cultures.  It  has  previously 
been  pointed  out  (p.  184)  that  the  cholera-vibrios  thrive 
especially  well  in  a  simple  alkaline  solution  of  peptone 
and  sodium  chlorid  (one  per  cent,  peptone  and  one-half 
per  cent,  sodium  chlorid).  It  may  be  added  that  the 
vibrios,  by  reason  of  their  motility  and  their  great  need  of 
oxygen,  tend  toward  the  surface  of  the  liquid  culture- 
medium,  and  there  undergo  enormous  multiplication. 
Upon  both  of  these  facts  is  based  the  method  of  peptone- 
culture,  which  has  the  further  great  advantage  that  it  leads 
to  the  desired  result  much  more  rapidly  than  the  plate- 
method.  A  platinum  loopful  of  the  suspected  feces,  or,  if 
this  be  obtainable,  a  flake  of  mucus,  is  introduced  into  a 
test-tube  or  an  Erlenmeyer  flask  containing  the  solution 
described,  and  the  vessel  is  exposed  in  the  thermostat  to  a 
temperature  of  37°  C.  (98.6°  F.).  As  soon  as  the  fluid 
exhibits  the  slightest  traces  of  turbidity,  which  usually 
occurs  in  the  course  of  from  six  to  ten  or  twelve  hours,  a 
specimen  is  taken  from  the  surface  and  is  examined  in 
hanging  drop  and  in  dry  cover-slip  preparations.  If  the 
examination  discloses  the  presence  of  a  pure  culture  of 
cholera-vibrios,  the  diagnosis  is  almost  certain.  In  most 
cases,  however,  the  procedure  is  not  quite  so  simple.  In 
the  superficial  layer  of  the  peptone-solution  the  vibrios  are 
usually  intermixed  with  other  microorganisms,  and  most 
frequently  with  the  bacterium  coli  commune.  There  then 
remains  nothing  but  to  make  plates  from  the  material  on 
the  surface.  These  plates,  however,  are  made  under  much 
more  favorable  conditions. 

There  is  now  no  longer  any  danger  that  the  small 
number  of  cholera-bacilli  that  were  present  originally  in 
the  feces  will  be  overrun  in  their  growth.  Through  the 
intermediation  of  the  peptone-culture  the  vibrios  have 
undergone  enormous  multiplication,  and  the   Petri  dishes 


ASIATIC   CHOLERA.  195 

now  exhibit,  in  consequence,  numerous  characteristic  colo- 
nies. 

From  the  gelatin-plates,  finally,  pure  cultures  are  made, 
with  which  the  cholera-red,  and  Gruber's  and  Pfeiffer's 
reactions  are  obtained,  and  experiments  on  animals  are 
undertaken.  When  all  these  yield  positive  results,  then 
the  diagnosis  of  cholera-bacilli  is  final  and  certain. 

Instead  of  gelatin-plates,  agar-plates  frequently  are  made. 
These  have  the  advantage  that  they  can  be  kept  in  the 
thermostat  at  a  temperature  of  37°  C.  (98.6°  F.),  and  can 
be  examined  after  from  eight  to  ten  hours.  It  has  already 
been  pointed  out  that  only  the  superficial  colonies  on  agar 
present  an  approximately  characteristic,  light  grayish- 
brown  appearance.  It  is,  therefore,  useful,  in  order  to  ob- 
tain only  such  superficial  colonies,  to  have  in  readiness 
agar-agar  poured  in  Petri  dishes,  upon  the  surface  of 
which  the  inoculating  material  (the  fertilized  peptone-cul- 
ture) is  smeared  by  means  of  a  platinum  loop.  As,  how- 
ever, the  agar-agar  always  expresses  a  certain  amount  of 
water  of  condensation,  which  renders  impossible  the  growth 
of  isolated  colonies,  it  is  necessary  to  place  dishes  into 
which  this  culture-medium  is  poured  in  the  thermostat  be- 
fore they  are  used,  until  the  fluid  has  evaporated,  or  they 
are  preserved  in  an  inverted  position.  It  is  not  sufficient 
in  making  a  diagnosis  to  find  light  grayish-brown,  trans- 
parent colonies,  but  by  means  of  preparations  it  must  be 
decided  whether  the  bacteria  constituting  the  colonies  cor- 
respond also  morphologically  with  cholera-vibrios. 

Examination  of  Water  for  Cholera-bacilli. — The  con- 
ditions are  somewhat  different  if  water  is  to  be  examined 
for  cholera-bacilli.  Formerly  it  was  the  custom  to  dilute 
freely  the  usually  much  polluted  water,  and  to  make  plates 
with  a  portion  of  a  drop.  Under  these  circumstances  it  was 
largely  a  matter  of  chance  whether  cholera-germs  were 
obtained  or  not.  It  would  be  necessary  for  them  to  be 
present  in  the  infected  water  in  considerable  number,  in 
order  to  be  demonstrated  in  this  way.  These  difficulties 
may  be  avoided  by  taking  large  amounts  of  water  for 
examination.  To  this  end  from  100  to  1000  cu.  cm. 
of  the  suspected  water  are  placed  in  sterilized  flasks, 
and  to  each  specimen  one  per  cent,  of  alkaline  peptone 
(preferably  the  peptone  of  Witte)  and  ^  of  one  per 
cent,  of  sodium    chlorid  are   added.      The   peptone    and 


196  CLINICAL  BACTERIOLOGY. 

sodium  chlorid  are  kept  in  readiness  in  sterilized  solutions, 
and,  for  instance,  5  cu.  cm.  and  2.5  cu.  cm.  of  20  per  cent, 
solutions  are  added  respectively  to  100  cu.  cm.  of  water. 
After  testing  its  alkalinity,  the  mixture  is  placed  in  the 
thermostat,  and  it  is  then  treated  in  exactly  the  same  way  as 
has  been  described  for  the  peptone -culture.  On  micro- 
scopic examination  of  the  surface  of  the  peptone  after  eight, 
ten,  fifteen,  and  twenty  hours,  curved  bacteria  that  closely 
resemble  the  comma-bacilli  are  found  in  many  samples  of 
water.  It  can  not  be  too  strongly  emphasized,  however,  that 
such  a  discovery  is  alone  not  demonstrative,  if  the  vibrios 
are  derived  directly  from  the  feces  of  a  sick  human  being. 
In  examinations  of  water  it  is  still  further  an  unavoidable 
postulate  that  plates  of  gelatin  or  of  agar  be  made  with  the 
fertilized  material  from  the  surface.  If  upon  these  are 
found  colonies  that  correspond  in  appearance  with  cholera- 
colonies,  it  is  not  yet  demonstrated  that  they  are  true 
comma-bacilli  and  not  merely  similar  microbes,  of  which  a 
number  have  already  been  described.  It  is  then  necessary 
to  make  pure  cultures,  and  these  are  to  be  identified  by 
means  of  the  indol-reaction,  by  experiments  on  animals, 
and  especially  by  means  of  Pfeiffer's  and  Gruber's  reactions 
(pp.  62,63,  187,  188). 

Disinfection  and  Prophylaxis. — Every  cholera-patient 
must  be  isolated  at  once.  Feces  and  vomited  matters,  as  well 
as  all  materials  soiled  therewith,  should  be  most  thoroughly 
disinfected.  The  details  of  disinfection  are  given  in  the  Ap- 
pendix. Those  who  come  in  contact  with  the  patient  should, 
further,  be  watched  with  care,  and  their  dejections  should  be 
examined  for  comma-bacilli.  Personal  prophylaxis  extends 
to  the  maintenance  of  the  digestive  tract  in  a  state  of  health 
(avoidance  of  all  dietetic  errors),  and  the  protection  against 
contamination  of  all  articles  of  food  (only  boiled  water  should 
be  drunk,  etc.).  The  general  prophylaxis  concerns  itself 
with  the  purification  of  the  water-supply  by  means  of  suit- 
able filtration,  with  the  protection  against  contamination  of 
waterways,  and,  above  all,  with  the  prevention  of  importation 
of  the  disease  from  abroad.  The  pilgrimages  to  Mecca  are 
under  the  scrutiny  of  the  International  Sanitary  Commission 
at  Alexandria.  Cholera  prevails  frequently  among  the 
pilgrims,  and  upon  the  slightest  suspicion  the  pilgrim-ships 
must  be  subjected  to  quarantine  before  being  permitted  to 
pass  through  the  Suez  Canal. 


ASIATIC  CHOLERA.  197 

Immunity. — A  large  number  of  persons — according  to 
Koch  almost  half — are  naturally  immune  to  cholera.  Re- 
covery from  one  attack  of  the  disease  confers  immunity  : 
at  least,  second  or  more  attacks  have  rarely  been  observed. 
Places  that  in  one  year  suffer  from  a  severe  visitation  of 
cholera  generally  remain  exempt  from  the  succeeding 
epidemic.  The  blood-serum  of  convalescents  from  cholera 
has,  in  some  instances,  exhibited  a  surprising  degree  of  pro- 
tective influence  for  guinea-pigs.  Thus  it  was  possible  to 
immunize  guinea-pigs  against  otherwise  fatal  inoculation 
with  cholera-vibrios,  in  one  case  by  means  of  o.Oi  cu.  cm., 
and  in  another  case  by  means  of  0.025  cu.  cm.  of  serum  from 
human  beings  that  had  recovered  from  cholera  (G.  Klemp- 
erer,  Lazarus).  As  Metschnikoff  has  shown,  not  the  serum 
of  all  convalescents  from  cholera  possesses  such  immu- 
nizing properties  ;  while,  on  the  other  hand,  these  may  be 
observed  occasionally  also  in  the  blood  of  persons  that 
have  died  of  cholera. 

Guinea-pigs,  rabbits,  and  goats  can  be  readily  immunized 
to  intraperitoneal  and  subcutaneous  infection  with  comma- 
bacilli  by  means  of  cultures  that  have  been  heated  to  a  tem- 
perature of  from  54°  C.  (129.2°  F.)  to  60°  C.  (140°  R), 
or  have  been  attenuated  by  any  other  means.  All  of  the 
methods  of  protective  inoculation  hitherto  employed  have, 
however,  been  efficient  only  with  relation  to  intraperitoneal 
or  subcutaneous  inoculation,  not  affording  certain  protec- 
tion against  introduction  of  the  vibrios  by  the  mouth. 
The  blood-serum  of  immunized  animals  is,  in  turn,  capable 
of  conferring  immunity.  In  the  process  of  preliminary 
treatment  the  bodily  fluids  of  the  animals  acquire  specific 
bactericidal  (lysogenic)  properties,  and  the  immunity  to  in- 
fection with  cholera-bacilli  appears  to  depend  exclusively 
upon  these  bactericidal  influences  (p.  62). 

According  to  the  view  of  R.  Pfeiffer,  the  blood-serum  of 
convalescents  from  cholera  likewise  contains  no  antitoxin, 
but,  on  the  other  hand,  the  same  lysogenic  substances  as 
the  serum  of  animals  immunized  artificially.  R.  Pfeiffer,  in 
consequence,  maintains  the  view  also  for  Asiatic  cholera  in 
human  beings  that  the  immunity  depends  upon  specific 
bactericidal  influences  (lysogenicity).  It  is  possible  in 
human  beings  to  stimulate  artificially  the  formation  of  the 
lysogenic  cholera  anti-bodies  in  the  blood.  It  is  only 
necessary  to  vaccinate  the  individuals  in  question  accord- 


198  CLINICAL  BACTERIOLOGY. 

ing  to  the  method  first  described  by  Haffkine.  This  ob- 
server injects  first  -^-^  of  an  agar-culture,  twenty-four 
hours  old,  carefully  devitalized  by  chloroform,  and  five  days 
later  -^^  of  a  living  virulent  culture,  and,  again,  after  the 
lapse  of  five  days,  yi  of  the  last.  Each  inoculation  is  fol- 
lowed by  an  insignificant,  slight,  painful  infiltration  and 
mild  fever,  which,  however,  soon  recede.  The  serum  of 
persons  thus  inoculated  acquires,  as  Kolle  has  shown, 
lysogenic  properties  at  least  equal  to  those  of  the  serum  of 
convalescents  from  cholera.  This  bactericidal  activity  be- 
gins on  the  fifth  day,  attains  its  maximum  on  the  twentieth 
day,  and  then  gradually  diminishes.  It  is,  however,  still 
demonstrable  after  the  lapse  of  a  year.  Kolle  has  shown, 
further,  by  experiments  on  human  beings,  that  a  single 
introduction  of  living  or  carefully  devitalized  vibrios  is  at- 
tended with  the  same  success,  so  that  repeated  inoculation 
is  not  at  all  necessary  to  attain  marked  bactericidal  activity. 
Little  of  a  positive  nature  can  yet  be  said  with  regard  to 
the  results  of  Hafifkine's  protective  inoculations  ;  but  they 
are  at  least  quite  encouraging. 


CHOLERA  NOSTRAS  AND  SUMMER  DIARRHEA. 

Cholera  nostras  includes  all  of  those  cases  of  severe 
diarrhea  presenting  symptoms  similar  to  those  of  Asiatic 
cholera,  but  with  an  absence  of  the  comma-bacilli  of  Koch 
from  the  feces,  and  without  epidemic  distribution.  In  by 
far  the  larger  number  of  cases  the  bacterium  coli  commune 
is  found  in  the  stools  and  in  the  vomited  matters.  Under 
these  conditions  the  bacterium  cpli  manifests  a  considerable 
degree  of  virulence  ;  and  in  cultures  from  a  case  of  cholera 
nostras  it  proves  far  more  malignant  in  experiments  on 
animals  than  in  cultures  from  normal  feces.  In  severe 
cases  of  cholera  nostras  the  bacterium  coli  is  often  present 
in  the  dejections  in  pure  culture.  In  some  cases  strepto- 
cocci have  been  found  in  the  feces  in  overwhelming  number, 
or  in  pure  culture,  and  in  isolated  instances  also  vibrios 
presenting  a  superficial  resemblance  to  Koch's  bacilli,  with- 
out, however,  agreeing  perfectly  with  these — as,  for  instance, 
the  vibrio  Lisbon,  in  Appendix. 

As  the  necessity  for  bacteriologic  examination  in  cases 
of  cholera  nostras  arises  only  in  the  presence  of  suspicious 


CHOLERA  NOSTRAS  AND  SUMMER   DIARRHEA.        199 

conditions,  when  the  point  to  be  determined  is  whether 
the  disease  in  question  is  true  Asiatic  cholera  or  not,  the 
mode  of  procedure  must  in  every  instance  be  precisely 
that  which  has  been  described  in  the  preceding  chapter  for 
Asiatic  cholera. 

Closely  related  to  cholera  nostras  is  the  so-called  sum- 
mer diarrhea  of  children.  The  probable  cause  of  this 
condition  is  thought  to  be  the  so-called  bacteria  of  bitter 
milk,  which  are  known  under  the  collective  name  of  bacillus 
lactis  Fliigge.  Fliigge  has  isolated  twelve  varieties  of  this 
organism,  all  of  which  belong  to  the  group  of  the  hay- 
bacillus,  and  with  which  they  have  many  points  of  resem- 
blance in  common.  (See  Hay-bacillus  and  Potato-bacillus 
in  Appendix.)  These  microorganisms  cause  peptonization 
of  the  casein  of  milk — hence  the  designation  peptonizing 
milk-bacteria — in  consequence  of  which  the  milk  acquires 
a  bitter  taste.  Some  of  them  give  rise  to  toxic  metabolic 
products,  which,  when  fed  to  young  dogs,  cause  diarrhea, 
muscular  weakness,  and  decline  of  temperature.  The 
whole  group  of  these  milk-bacteria  is  characterized  by  the 
formation  of  highly  resistant  spores  that  withstand  boiling 
for  several  hours  without  injury.  It  is  on  this  account  that 
the  sterilization  of  milk  is  attended  with  so  much  difficulty  ; 
even  the  method  of  Soxhlet  is  incapable  of  destroying  the 
peptonizing  microorganisms.  If  such  an  insufficiently  or,  to 
use  the  common  expression,  partially  sterilized  milk  is  pre- 
served at  a  moderate  temperature — as,  for  instance,  22°  C. 
(71.6°  F.)  or  above — the  spores  develop,  and  the  poisons 
mentioned  are  generated.  For  this  reason  great  care  must 
be  taken  that  milk  prepared  by  the  Soxhlet  method  is  kept 
in  a  cool  place  until  shortly  before  it  is  used,  and  in 
summer  best  in  a  refrigerator.  The  custom  of  sending 
with  children  a  supply  of  milk  for  a  considerable  time, 
heated  and  kept  in  special  warming  bags,  should  be  aban- 
doned, as  in  the  presence  of  numerous  spores  germination 
and  beginning  multiplication  may  set  in  speedily  (in  the 
course  of  one  or  two  hours).  These,  however,  are  to  be 
considered  as  dangerous,  as,  according  to  the  investigations 
of  Liibbert,  *'  the  active  principle  of  the  decomposed  milk 
is  to  be  looked  for  in  the  bodies  of  the  bacilli."  The 
anaerobic  microorganisms  to  be  found  in  milk — the  bacillus 
butyricus  Botkin  and  that  of  Fliigge — probably  play  no 
part  in   the   etiology  of  the  intestinal  catarrh  of  infants. 


200 


CLINICAL  BACTERIOLOGY. 


They  occur  only  in  small  number  and  change  the  appear- 
ance and  the  odor  of  the  milk  to  such  a  degree  that  it  will 
probably  not  be  used. 


PLAGUE. 

The  exciting  agent  of  plague  was  discovered  by  Kitasato 
and  Yersin  during  an  epidemic  of  cholera  in  China  in  the 
year  1894. 

Morphology. — The  plague-bacillus  is  a  small,  nonmotile  rod, 
with  rounded  extremities.  It  is  believed  to  possess  a  capsule, 
which,  however,  is  not  readily  demonstrated.  Plague-bacilli 
vary  extraordinarily  in  form,  both  in  cultures  and  in  the 
products   of  the  disease  in  human  beings  and   animals.      At 


Fig.  50. — Bacillus  of  bubonic  plague  (Yersin). 


times  they  appear  as  short,  compact  bacteria,  at  other  times  they 
are  suggestive  of  diplococci,  distinct  rods,  short,  sharply  bent 
threads,  and,  finally,  in  older  cultures,  degeneration-products 
occur  in  the  form  of  swollen  spheres  and  clubs.  The  plague- 
bacillus  does  not  stain  by  Gram's  method,  but,  on  the  other 
hand,  readily  with  all  aniline  dyes,  and  most  strikingly  with 
methylene-blue.  Under  these  circumstances  it  exhibits  distinct 
polar  staining.  The  extremities  are  more  deeply  stained  than 
the  central  portion,  which  after  feeble  action  of  the  staining 
solution  appears  to  be  a  deficiency. 

Spores  are  not  present. 

The  temperature-optimum  is  that  of  the  body,  although  the 
plague-bacillus  thrives  well  at  room-temperature. 


PLAGUE.  201 

Appearance  in  Culture. — On  gelatin-plates  the  colonies 
appear  as  finely  granular  bodies  of  brownish  color,  with  a 
smooth  border.  The  superficial  colonies  possess  a  delicate  mar- 
ginal zone.     The  culture-medium  is  not  liquefied. 

In  gelatin  stab-cultures  slow ,  uniform  growth  takes  place  along 
the  line  of  inoculation,  while  a  smooth  deposit  forms  upon  the 
surface. 

In  gelatin  streak-cultures  a  light  yellowish  deposit  forms. 

On  agar-plates,  after  twenty-four  hours,  delicate,  dewdrop-like 
colonies  form  that  appear,  after  the  lapse  of  forty-eight  hours, 
as  grayish  points,  whose  border  is  slightly  iridescent.  At  times 
a  number  of  large  colonies  are  seen  among  the  smaller. 

In  agar  stab-cultures  a  tough,  mucoid  coating  develops,  the 
water  of  condensation  is  rendered  turbid,  but  no  membrane  forms. 

In  Loffler' s  blood-serum  the  same  development  takes  place  as 
in  agar  streak-cultures. 

Bouillon  is  rendered  diffusely  turbid.  If,  however,  it  be  in- 
oculated with  a  coherent  bacterial  mass  from  an  agar-culture, 
the  bacilli  develop  at  the  bottom  of  the  tube,  while  the  over- 
lying fluid  remains  clear.  In  this  way  a  growth  is  obtained  that 
is  suggestive  of  streptococci. 

Milk  is  a  poor  culture-medium,  and  is  not  coagulated. 

Upon  potatoes  a  scanty,  whitish-gray  coating  forms  at  a  tem- 
perature of  37°  C.  (98.6°  F.). 

In  culture-media  containing  sugar  the  plague-bacillus  does 
not  generate  gas.  It  does  not  form  indol  either  in  bouillon  or  in 
peptone-water. 

Wladimiroff  and  Kressling  believe  a  neutral  reaction  of  the 
culture-medium  most  favorable  for  growth.  Addition  of  glycerin 
to  the  nutrient  medium  is  rather  disadvantageous. 

Vital  Capability  of  Plague-bacilli. — Exposure  for  ten 
minutes  to  a  temperature  of  55°  C.  (131°  F. )  or  for  five  min- 
utes to  a  temperature  of  80°  C.  (176°  F.)  suffices  to  cause  the 
death  of  the  bacilli.  They  are  destroyed  at  once  by  a  i  :  1000 
solution  of  mercuric  chlorid,  and  in  ten  minutes  by  one  per 
cent,  carbolic  acid  or  one  per  cent,  lysol.  Mineral  acids  are 
very  active  :  Sulphuric  acid,  i  :  2000,  destroys  the  bacilli  within 
five  minutes ;  hydrochloric  acid,  i  :  1000,  within  thirty  min- 
utes. When  material  containing  plague-bacilli  was  transferred 
to  linen,  wool,  earth,  etc.,  the  longest  period  for  which  life  was 
preserved  was  eight  days.  The  same  observation  was  made  with 
regard  to  preserved  portions  of  organs.  The  sputum  from 
cases  of  pneumonia  complicating  plague,  kept  in  tubes  closed 
with  cotton,  was  no  longer  infective  after  sixteen  days.  In  or- 
dinary tap-water  the  bacilli  die  in  three  days,  in  sterilized  water 
in  eight  days,  in  sterilized  bilge-water  after  five  days  (German 
Plague-commission). 


202  CLINICAL  BACTERIOLOGY. 

Portals  of  Infection  for,  and  Mode  of  Distribution  of, 
Plague-bacilli. — The  plague-bacillus  gains  entrance  into  the 
human  organism  most  frequently  by  way  of  slight  injuries, 
scratch-wounds,  etc.  Infection  through  the  skin  may  take 
place  simultaneously  at  several  different  points  of  the  body  in 
the  same  case ;  it  has  not  yet  been  decided  whether  it  can 
occur  through  the  intermediation  of  insects.  The  related 
lymph-glands  nearest  to  the  portal  of  infection  become 
swollen,  most  frequently  those  of  the  inguinal  and  axillary 
regions  (primary  plague-boils).  Sometimes  the  glands  of 
the  first  degree  remain  free  or  are  but  slightly  irritated,  and 
only  those  of  the  second  and  third  degrees  are  seriously 
affected.  Sometimes  the  entire  chain  of  glands  from  the 
nearest  to  the  most  remote  are  intensely  inflamed.  At  the 
site  of  infection  a  pustule  or  a  carbuncle  is  not  rarely  found, 
and  between  this  and  the  related  lymphatic  enlargement 
frequently  a  distinct  lymphangitis  can  be  traced.  In  the 
milder  cases  the  buboes  disappear.  If,  however,  the  gland- 
filter  is  broken  through,  the  plague-bacilli  gain  entrance 
through  the  glands  into  the  blood  and  the  internal  organs, 
and  the  condition  of  plague-septicemia,  which  almost  always 
terminates  fatally,  is  established.  If  the  buboes  undergo 
suppuration,  the  plague-bacilli  usually  die  quickly.  Some- 
times, however,  secondary  infection  with  streptococci  is 
superadded,  and  this  then  constitutes  a  serious  menace  to 
the  patient.  In  the  septicemic  form  of  the  disease  the  bacilli 
gain  entrance  into  the  feces  and  the  urine  in  consequence  of 
the  hemorrhages  that  occur  so  frequently. 

A  second  and  less  common  portal  of  infection  is  consti- 
tuted by  the  lungs.  Bronchopneumonic  foci  occur,  in 
which  the  exciting  agents  of  plague  are  found  in  pure  cul- 
ture or  in  association  with  diplococci  and  streptococci.  "The 
sputum  of  such  patients  contains  the  plague-bacillus. 
Finally,  cases  of  primary  infection  through  the  tonsils 
have .  been  observed,  rapidly  giving  rise  to  general  infec- 
tion. 

The  German  Commission  that  went  to  Bombay  in  the 
beginning  of  the  year  1897  for  the  study  of  plague,  and 
from  whose  report  the  foregoing  statements  are  taken,  em- 
phasized that  the  incredible  filth  amid  which  the  natives 
live,  their  crowding  together  in  small  dwellings,  the  frequent 
small  injuries,  especially  of  the  bare  feet,  the  constant 
scratching  induced  by  vermin,  are  quite  sufficient  to  explain 


PLAGUE.  203 

the  frightful  frequency  with  which  plague  prevails  among 
the  lower  classes  in  Bombay. 

Plague  adheres  obstinately  to  human  habitations.  It 
does  not  extend  in  an  explosive  manner  over  large  portions 
of  the  same  city,  but  passes  from  house  to  house.  In  addi- 
tion to  human  intercourse,  an  important  part  is  played  by 
the  remarkable  relations  that  exist  between  rats  and  simi- 
lar vermin  and  bubonic  plague,  as  was  already  recognized  in 
the  middle  ages.  From  numerous  sources  the  German 
Plague-commission  was  informed  that  the  outbreak  of  the 
epidemic  was  preceded  by  a  pestilential  disease  among  rats, 
with  an  enormous  mortality.  The  natives  believe  so  firmly 
in  the  connection  between  the  plague  of  rats  and  that  of 
human  beings  that  some  of  them  will  at  once  leave  their 
houses  if  they  find  a  dead  rat  present.  After  great  effort 
the  German  Plague-commission  succeeded  in  obtaining  the 
fresh  cadaver  of  a  rat  that  had  been  infected,  and  in  it  they 
found  numerous  plague -bacilli.*  Yersin  has  made  the  note- 
worthy statement  that  he  found  the  plague-bacillus  in  the 
dust  and  the  filth  of  plague-houses. 

Experiments  on  Animals. — The  animal  most  suscepti- 
ble to  plague  is  the  rat.  Minimal  amounts  of  a  culture 
sufifice  on  cutaneous  inoculation  to  cause  death  regularly. 
The  same  result  may  be  attained  by  application  of  the 
plague-bacilli  to  the  mucous  membrane  of  the  eye  or  of  the 
nose.  Rats  die  after  eating  quite  small  amounts  of  plague- 
infected  food,  or  after  gnawing  the  bodies  of  other  rats  dead 
of  plague.  The  latter  circumstance  especially  is  of  great  im- 
portance. It  explains  the  incredibly  rapid  spread  of  rat- 
plague,  and  also  renders  it  probable  that  rats  are  responsible 
for  the  extension  of  the  disease  from  house  to  house. 
Next  to  the  rat  in  susceptibility  to  the  plague  is  the  gray 
ape.  Plague-bacilli  are  pathogenic  for  the  usual  labora- 
tory-animals in  general ;  pigeons  alone  form  an  exception. 
At  the  autopsy  a  mucous,  at  times  hemorrhagic,  exudate 
is  found  at  the  point  of  injection.  The  nearest  glands  are 
most  enlarged,  and  they  undergo  suppuration  but  seldom. 
The  blood  and  the  internal  organs  contain  the  bacilli,  the 
former,  however,  in  but  small  amount.  Kolle  calls  atten- 
tion to  a  more  protracted  course  for  the  disease,  which 
takes  place  if  rats  and  guinea-pigs  are  inoculated  with  cul- 

*  It  is  believed  that  squirrels  and  monkeys  also  may  be  infected  by  plague 
and  act  as  disseminators  of  the  disease. — A.  A.  E. 


204  CLINICAL   BACTERIOLOGY. 

tures  that  have  already  been  carried  on  artificially  for  sev- 
eral years.  The  animals  then  die  in  the  course  of  the 
second  week,  and  one  or  more  glands,  up  to  the  size  of  a 
walnut,  are  found,  which  contain  creamy  pus  in  their  cen- 
tral portions,  in  which  plague-bacilli  are  present  in  large 
numbers.  An  involuntary  experiment  in  the  human  being 
is  also  on  record  :  A  member  of  the  German  Plague-com- 
mission suffered  infection  in  making  an  autopsy  of  a  body 
dead  of  plague.  Two  days  later  a  small  pustule  formed  on 
the  right  hand,  followed  shortly  by  a  lymphangitis,  and  a 
swollen  gland  in  the  axilla.  The  discharge  from  the  pus- 
tule contained  plague-bacilli.  In  spite  of  the  alarming 
character  of  the  disease  at  first,  recovery  took  place. 

Etiologic  Relations  of  the  Bacilli  to  Plague. — The  con- 
stant presence  in  all  cases  of  plague  and  the  positive  out- 
come of  experiments  on  animals  indicate  with  certainty  that 
the  plague-bacillus  is  the  specific  exciting  agent  of  plague. 
In  accordance  with  what  has  been  said,  the  disease  is  to  be 
looked  upon  rather  as  an  infectious  disease  ;  but  a  toxic 
element  is  by  no  means  wanting.  The  hyperemia  and  the 
ecchymoses  of  the  stomach  and  the  bowel  are  considered 
by  the  German  Plague-commission  as  purely  toxic  effects. 
As  an  evidence  of  the  toxic  action,  the  case  is  reported  of 
a  fetus  born  on  the  third  day  of  the  disease  in  the  mother, 
in  which  the  characteristic  hemorrhages  were  found,  al- 
though all  portions  of  the  body  were  free  from  germs. 
Certain  sequelae  also  are  to  be  attributed  to  the  toxic 
effects  :  paralysis  of  the  recurrent  laryngeal  nerve,  amauro- 
sis, aphasia,  deafness,  paraplegia,  etc.  The  poison  of  the 
plague-bacilli  is  probably  contained  within  the  bodies  of 
the  bacteria.  The  German  Commission  dried  cultures 
carefully,  mixed  them  with  water,  and  heated  them  to  a 
temperature  of  65°  C.  (149°  F.).  As  much  as  eighty 
milligrams  of  the  dry  residue  were  introduced  into  the 
peritoneal  cavity  of  brown  apes,  which  exhibited  only  mild 
manifestations  of  disease,  slight  decline  of  temperature, 
want  of  energy,  and  anorexia.  In  brown  apes,  which  are 
by  no  means  so  susceptible  to  plague  as  gray  apes,  this 
toxic  effect  is,  therefore,  but  little  marked. 

Bacteriologic  Diagnosis  of  Plague. — The  suppurating 
plague-buboes  contain  the  characteristic  bacteria  in  enor- 
mous number.  These  are  readily  recognized  by  their  polar 
staining    when    treated    with    methylene-blue.      A  similar 


PLAGUE.  205 

phenomenon  is  exhibited  among  the  pathogenic  micro- 
organisms only  by  the  bacilli  of  chicken-cholera.  These, 
however,  are  larger  than  the  plague-bacilli,  and  are  of 
absolutely  no  significance  with  relation  to  human  beings.  If 
suppuration  has  not  yet  taken  place,  the  diagnosis  is  not  so 
simple,  although  it  appears  most  necessary  just  under  these 
conditions.  Puncture  of  the  buboes  for  this  purpose  was 
considered  by  the  Commission  in  its  first  communication 
as  not  unattended  with  danger  on  account  of  the  possibility 
of  opening  a  blood-vessel.  It  has,  however,  since  been 
shown  that  English  physicians  in  plague-hospitals  made 
long  incisions  into  the  diseased  glands,  with  subsequent 
antiseptic  treatment,  for  therapeutic  purposes.  In  connec- 
tion with  this  procedure  fluid  from  the  gland  is  readily 
obtained  for  the  purpose  of  making  cover-slip  preparations, 
plates,  and  cultures. 

Microscopic  examination  of  the  blood  yields  successful 
results  only  in  cases  of  general  infection.  Cultural  investi- 
gation of  the  blood,  however,  yields  better  results.  In 
order  to  cultivate  plague-bacilli  from  sputum,  and,  in  general, 
from  bacterial  mixtures,  it  is  best  to  make  gelatin  streak- 
cultures.  The  suspected  material  is  spread  in  several 
streaks  upon  the  surface  of  a  solidified  gelatin-plate.  The 
plague-bacilli  grow  well  at  a  temperature  as  low  as  from 
22°  C.  (71.6°  F.)  to  25°  C.  {jj''  F.) ;  while  the  associated 
bacteria — the  diplococcus  lanceolatus  and  the  streptococcus 
— grow  but  feebly  if  at  all.  From  a  diagnostic  point  of 
view  it  is  further  of  the  greatest  significance  that  the  blood- 
serum  of  human  beings  and  of  animals  that  have  recovered 
from  infection  with  plague  possesses  agglutinating  activity 
(German  Commission).  It  is  said  that  the  serum  does  not 
cause  agglutination  before  the  second  week,  and  that  this 
is  most  marked  in  the  second  and  third  weeks. 

In  cases  of  mixed  infection,  which  occur  especially  in 
conjunction  with  suppuration  of  the  buboes,  streptococci 
appear,  not  only  in  the  glands,  but  also  in  the  blood. 

The  prophylaxis  of  plague  consists  in  disinfection 
of  all  products  of  the  disease  that  contain  the  specific 
bacillus.  In  the  first  place,  general  hygienic  precautions 
should  be  observed,  with  especial  regard  to  cleanliness  and 
ventilation  of  dwellings,  care  of  the  skin,  etc.  In  general, 
plague  attacks  preferably  the  lower  classes  of  society,  ex- 
posed   to   want,   filth,    and   misery.      Isolation  of  plague- 


206  CLINICAL  BACTERIOLOGY. 

patients  and  observation  of  those  b^^  whom  they  are  sur- 
rounded are  naturally  necessary. 

Immunity  and  Protective  Inoculation. — Plague-bac- 
teria retain  their  virulence  until  shortly  before  death.  It  is, 
therefore,  impossible  to  immunize  susceptible  animals  with 
old  cultures  (German  Commission).  Slightly  susceptible 
animals  can  be  readily  immunized  by  increasing  doses  of 
living  bacilli.  Highly  susceptible  animals  must,  however, 
be  treated  carefully  with  dead  cultures.  For  this  purpose 
it  is  best  to  employ  cultures  that  have  been  exposed  for  an 
hour  to  a  temperature  of  65°  C.  (149°  F.).  In  subjecting 
a  brown  ape  to  protective  inoculation  the  Commission  em- 
ployed a  heated  agar  streak-culture.  Seven  days  later  the 
animal  was  immune  to  subcutaneous' infection,  but  only  after 
it  had  recovered  from  this  was  it  protected  against  intra- 
peritoneal inoculation.  Filtered  bouillon-cultures  possess 
only  slight  immunizing  properties. 

Haffkine  has  employed  his  method  of  protective  inocu- 
lation, which  he  first  recommended  for  cholera,  also  in  the 
treatment  of  plague.  A  fluid  preparation,  made  from  care- 
fully devitalized  plague-bacilli,  is  injected  in  doses  of  from  ^ 
to  2j4  cu.  cm.,  in  accordance  with  the  age  of  the  individuals. 
Slight  reaction  follows,  and  the  injection  is  repeated  in  a 
somewhat  larger  dose  after  the  lapse  of  eight  or  ten  days. 
The  results  obtained  with  Haff  kine's  method  are  not  un- 
favorable, but  a  definite  decision  as  to  its  value  must  be  de- 
ferred for  the  present ;  at  any  rate,  the  protection  conferred 
is  not  absolute. 

A  plague-serum  is  prepared  in  the  Pasteur  Institute  for 
therapeutic  purposes  from  the  blood  of  highly  immunized 
horses.  In  the  observations  of  the  German  Commission, 
three  cubic  centimeters  of  this  serum  sufficed  to  protect 
brown  apes  against  subsequent  subcutaneous  infection.  As 
much  as  ten  cubic  centimeters,  however,  did  not  suffice  to 
protect  susceptible  gray  apes.  The  serum  exhibits,  also, 
an  undoubtedly  curative  action  (after  twelve  hours)  in  pre- 
viously inoculated  brown  apes.  With  regard  to  its  efficacy 
in  cases  of  plague,  nothing  definite  can  as  yet  be  stated. 


DIPHTHERIA.  207 


DIPHTHERIA. 

The  diphtheria-bacillus  was  grown  in  pure  culture  in 
1884  by  Loffler. 

The  diphthej'ia-bacillus  is  a  rather  plump  rod  of  varying  size, 
from  I  to  6  /^  long  and  from  0.5  to  i  ix  thick.  Its  form  is  sub- 
ject to  great  variations  in  different  cultures.  At  times  it 
appears  as  a  small,  wedge-shaped  body ;  at  other  times  as  a 
rather  long  body,  with  a  bulbous  thickening  at  one  extremity — 
a  so-called  club  ;  and  at  still  other  times  as  a  double  club,  or  a 
dumb-bell.  Slender  forms,  occasionally  curved  slightly,  are  also 
observed,  especially  in  membranes.  Not  rarely  bifurcations  are 
encountered,  and  upon  the  basis  of  this  observation  diphtheria- 


c^  ^/-^'^ 


>>1£    «^'  K 


Fig.  51. — Bacillus  diphtlieriae  from  a  pure  culture  (Stengel). 

bacilli  have  been  placed  in  relation  with  streptothrices,  and 
even  with  hyphomycetes.  The  diphtheria-bacillus  is  incapable 
of  independent  movement. 

Spore -formation  is  wanting. 

Staining  Properties. —  The  diphtheria-bacillus  is  best 
stained  with  methylene-blue  or  dilute  carbolfuchsin.  Gentian- 
violet  overstains  and  conceals  the  more  delicate  structural  rela- 
tions. The  organism  is  stained  by  Gram's  method.  The  small, 
blunt  clubs  stain  equably,  while  the  longer  specimens  exhibit 
unstained  areas,  so  that  the  rods  appear  in  places  to  consist  of 
several  segments.  Fliigge  has  described  a  method  of  double 
staining,  devised  by  M.  Neisser,  which  is  considered  to  possess 
essential  differential  diagnostic  significance.  The  preparation 
is  placed  for  from  three  to  five  seconds  in  an  acetic-acid  solu- 
tion of  methylene-blue  (methylene-blue  i,  alcohol  20,  distilled 
water  950,  glacial  acetic  acid  50),  rinsed  in  water,  and  counter- 
stained  for  from  three  to  five  seconds  in  vesuvin  (vesuvin  2, 


208 


CLINICAL  BACTERIOLOGY. 


dissolved  in  boiling  distilled  water  loco,  and  filtered).  The 
bodies  of  the  bacilli  appear  brown,  and,  as  a  rule,  they  contain 
two  blue  granules,  which  have  at  once  been  intensely  stained  by 
the  first  aniline  dye,  and  have  not  yielded  their  color  to  the 
vesuvin  subsequently  employed.  These  are  the  so-called  Babes- 
Ernst  bodies  (p.  19).  The  form,  arrangement,  and  situation  of 
these  bodies  are  considered  as  characteristic  of  the  diphtheria- 
bacillus  under  the  following  conditions :  The  preparations 
must  be  made  only  from  cultures  that  have  grown  upon  Loffler's 
blood-serum  (solidified  at  100°  C. — 212°  F. )  at  a  temperature 
of  34°  C.  (93.2°  F. )  or  35°  C.  (95°  F. ),  and  never  above 
36°  C.  (96.8°  F.).     The  cultures  must  not  be  less  than  nine 


Fig.  52. — Bacillus  diphtheriae,  from  a  culture  upon  blood-serum ;    X  looo  (Frankel 

and  Pfeiffer). 


hours,  and  not  more  than  from  twenty  to  twenty-four  hours,  old. 
At  one  end,  more  frequently  at  both  ends,  of  the  brown  rod  a 
blue  granule  is  then  to  be  seen,  and  not  rarely  a  third  is  visible  in 
the  middle.  These  granules  are  oval  in  shape,  and  possess  a 
greater  diameter  than  the  bacillus  itself,  which,  however,  if  the 
whole  appearance  is  to  be  considered  of  diagnostic  significance, 
must  be  distinctly  visible  in  its  entire  length  and  form. 

It  is  further  said  to  be  characteristic  of  the  diphtheria-bacillus 
that  the  individual  bacteria  are  arranged  side  by  side  like  pali- 
sades. As  the  most  distinctive  feature,  M.  Neisser  considers 
the  appearance  of  impression-preparations  from  a  serum-plate 
six  hours  old  that  has  developed  at  a  temperature  of  from  34° 
C.    (93.2°   F.)  to   36°   C.    (96.8°  F.).      In  these  there  are 


DIPHTHERIA.  209 

visible  **  moderate-sized  free  masses  in  which  the  slender,  rather 
long,  slightly  curved  bacilli  lie  in  characteristic  irregular  ar- 
rangement— an  appearance  that  is  to  some  degree  represented 
by  placing  the  extended  fingers  of  one  hand  in  varying  combi- 
nations over  or  by  the  side  of  those  of  the  other." 

Cultural  Properties. — The  diphtheria-bacillus  thrives  only 
at  temperatures  between  20°  C.  (68°  F.)  and  40°  C.  (104°  F.) 
upon  all  slightly  alkaline  culture-media.  Its  temperature-opti- 
mum is  that  of  the  body. 

Upon  gelatin-plates  round,  whitish  colonies  form  that  remain 
small.  Microscopically  these  appear  yellowish  brown  and  gran- 
ular, with  an  irregular  border. 

In  gelatin  stab-cultures  similar  small,  whitish,  spherical  colo- 
nies, not  exceeding  a  certain  size,  form  along  the  line  of  inocu- 
lation. At  a  temperature  of  24°  C.  (75.2°  F. )  superficial 
growth  takes  place,  with  indications  of  nail-culture.  The  gela- 
tin is  not  liquefied. 

Upon  agar^  and  better  upon  glycerin-agar  plates,  after  from 
twenty-four  to  forty-eight  hours,  small,  grayish-white,  glistening 
colonies  form  that  often  exhibit  microscopically  concentric  lami- 
nation ;  with  low  powers  of  the  microscope  they  appear  pecu- 
liarly granular,  with  an  irregular  border. 

In  agar  streak-cultures,  after  twenty-four  hours,  small,  trans- 
lucent, slightly  raised  colonies  appear.  Further  growth  is  in- 
considerable, and  scarcely  extends  beyond  the  line  of  inocula- 
tion. 

In  agar  stab -cultures  the  colonies  develop  along  the  line  of 
inoculation,  and  there  is  slight  growth  upon  the  surface. 

Upon  Loffler  s  serum,  after  twenty-four  hours,  fairly  large, 
whitish,  opaque  colonies  of  firm  consistence  appear.  In  the 
succeeding  days  these  colonies  increase  but  little  in  size. 
Loffler's  blood-serum  (p.  84)  constitutes,  all  in  all,  the  best 
culture-medium  for  diphtheria-bacilli.  It  is,  therefore,  always 
employed  for  purposes  of  differential  diagnosis. 

In  bouillon,  after  twenty-four  hours  some  precipitate  has  formed, 
after  two  days  slight  turbidity,  which  increases  to  the  fifth  day, 
then  to  grow  less,  until  finally  the  fluid  overlying  the  crumbling, 
flocculent  precipitate  is  completely  clear.  Not  rarely  a  thin, 
fragile  coating  appears  upon  the  surface  of  the  bouillon. 

A  delicate  coating  forms  upon  the  surface  of  potato  rendered 
alkaline. 

Milk  constitutes  a  favorable  nutritive  medium,  but  is  not 
coagulated. 

In  boiled  and  unboiled  egg,  both  white  as  well  as  yolk,  the 
diphtheria-bacillus  grows  well;  upon  coagulated  egg-albumin 
it  not  rarely  exhibits  branching. 

In  slightly  alkaline  Loffler's  meat-infusion  bouillon  the  diph- 
14 


210  CLINICAL  BACTERIOLOGY. 

theria-bacillus  generates  acid.  The  increase  in  acidity  is  dis- 
tinctly appreciable  within  twenty-four  hours  ;  it  augments  from 
the  second  day,  then  to  subside.  Later,  after  two  or  three  weeks, 
the  bouillon  becomes  again  alkaline.  On  addition  of  litmus  to 
the  culture-medium  these  variations  can  be  distinctly  followed 
by  the  changes  in  color. 

Tenacity  of  the  Diphtheria-bacilli. — A  solution  of 
mercuric  chlorid,  i  :  looo,  destroys  cultures  in  a  thick 
layer  within  twenty  seconds  ;  and  five  per  cent,  potassium 
permanganate,  five  per  cent,  aqueous  solution  of  carbolic 
acid,  three  per  cent,  solution  of  carbolic  acid  in  thirty  per 
cent,  alcohol,  four  per  cent,  solution  of  kresol  in  forty 
per  cent,  alcohol,  within  the  same  time.  Five  per  cent, 
potassium  chlorate  is  still  ineffective  after  sixty  seconds 
(Loffler).  Pure  lemon-juice  likewise  destroys  the  bacilli 
speedily.  They  are  destroyed  by  exposure  for  ten  minutes 
to  a  temperature  of  60°  C.  (140°  F.),  although  in  a  some- 
what thicker  layer  they  withstand  drying  for  months.  In 
the  form  of  dust,  however,  they  rapidly  die.  They  with- 
stand cold  well,  although  in  the  refrigerator  the  diphtheria- 
bacilli  rapidly  lose  their  property  of  generating  toxins. 
Abel  found  diphtheria-bacilli  on  building-blocks  with  which 
a  child  suffering  from  diphtheria  had  played  six  months 
previously.  Diphtheria-bacilli  have  been  found,  further, 
upon  soiled  bed-linen,  on  the  rim  of  a  drinking-glass, 
in  the  hair  and  on  the  shoes  of  nurses,  etc.  In  gelatin- 
cultures  they  may  survive,  according  to  Loffler,  for  three 
hundred  and  thirty-one  days.  Diphtheric  membranes  dried 
and  preserved  in  the  dark  yield  cultures  even  after  the 
lapse  of  months. 

Pathogenic  Properties  of  the  Diphtheria-bacillus  for 
Animals. — Diphtheria  does  not  naturally  occur  in  animals. 
So-called  spontaneous  diphtheria  of  fowl,  of  pigeons,  etc., 
are  etiologically  different  diseases. 

Diphtheria-bacilli  give  rise  to  the  formation  of  true  diph- 
theric pseudoniembrane ,  with  multiplication  of  the  bacilli, 
in  the  previously  injured  vagina  or  conjunctiva  of  guinea- 
pigs,  in  the  trachea  of  guinea-pigs  and  rabbits  after  trache- 
otomy. Most  birds,  especially  pigeons  and  chickens,  then 
young  dogs,  rabbits,  and  especially  guinea-pigs,  are  suscep- 
tible to  the  diphtheria-bacillus.  Subcutaneous  introduction 
of  the  bacilli  is  first  followed  by  purely  local  alterations  : 
more  or  less  extensive  hemorrhagic  edema  of  the  subcuta- 


DIPHTHERIA.  211 

neous  connective  tissue.  Then,  in  the  presence  of  fever, 
there  develop  pleuritic  effusions,  swelling  and  redness  of 
the  adrenal  glands,  hemorrhages  into  the  structure  of  the 
lymphatic  glands,  and  death,  preceded  by  decline  of  the 
temperature,  occurs  in  the  course  of  from  twenty-four  to 
forty-eight  hours.  If  the  virulence  of  the  bacillus  is  slight, 
the  disease  is  protracted  :  it  may  last  three,  four,  or  five 
days,  and  even  several  weeks.  In  the  less  susceptible  rabbit 
the  slow  course  is  the  rule.  Under  these  circumstances 
there  occur  true  diphtheric  palsies,  at  first  involving  the 
posterior  extremities  of  the  animal,  then  the  anterior,  and 
finally  the  muscles  of  the  neck.  Death  takes  place  after 
marked  emaciation.  At  the  autopsy  fatty  degeneration  of 
the  liver  and  the  kidneys  is  found,  and  also  inflammatory 
changes  in  the  spinal  cord  and  the  nerves.  If  the  virulence 
is  still  further  diminished,  the  constitutional  symptoms 
(fever)  and  the  remote  manifestations  (pleuritis,  palsies)  are 
less  conspicuous.  After  recovery  from  the  local  inflamma- 
tory process,  which  terminates  in  necrosis  of  the  skin,  the 
animal  may  be  restored  to  health. 

Physiology  of  the  Disease  in  Animals. — Whether 
their  virulence  be  great  or  slight,  the  bacilli  remain  at  the 
site  of  introduction,  and,  as  a  rule,  they  do  not  penetrate 
more  deeply  into  the  organism.  Multiplication  of  the 
bacilli  at  the  point  of  injection  takes  place  only  to  a  small 
extent,  and  only  at  the  beginning — in  the  first  six  or  eight 
hours  ;  later,  the  number  of  bacteria  rather  diminishes. 
They  may,  however,  survive  for  a  long  time,  and  they  have 
been  found  alive  after  weeks  among  the  necrotic  portions 
of  skin  at  the  point  of  injection.  The  constitutional  dis- 
ease of  animals  results  exclusively  from  the  poison  gene- 
rated by  the  bacteria :  the  diphtheria-toxin.  The  disease 
developed  in  animals  by  diphtheria-bacilli  is,  thus,  a  purely 
toxic  one.  If  the  poison  alone,  without  the  bacilli,  is  intro- 
duced into  the  bodies  of  animals,  there  result  the  same 
morbid  manifestations,  with  the  exception  of  the  false 
membrane,  and  especially  the  palsies,  just  as  after  inocula- 
tion with  the  bacilli  themselves.  In  experimental  intoxi- 
cation of  animals  a  bouillon-culture,  several  weeks  old,  is 
employed  for  inoculation  whose  reaction,  originally  alka- 
line, has  become  acid  and  is  again  rendered  alkaline,  and 
which  has  been  freed  of  bacteria  by  filtration  through  a 
porcelain  filter  (Chamberland  filter  in  which  the  fluid  con- 


212  CLINICAL  BACTERIOLOGY. 

taining  the  bacteria  is  under  positive  pressure,  or  Kitasato's 
porcelain  cylinders  in  which  suction  of  the  filtrate  takes 
place).  Instead  of  filtering  them,  the  bacilli  in  the  culture 
may  be  destroyed  by  addition  of  carbolic  acid  in  propor- 
tion of  o.  5  per  cent. 

Reference  has  been  made  to  the  efforts  directed  to 
obtaining  the  poison  chemically  pure  from  the  toxin-con- 
taining bouillon-filtrate  (p.  29).  Brieger  and  Boer  have 
made  the  greatest  advances  in  the  preparation  of  the  diph- 
theria-toxin. To  the  toxin-containing  filtrate  they  add  twice 
as  much  of  a  one  per  cent,  solution  of  zinc  chlorid.  The 
resulting  precipitate  is  agitated  with  from  a  three  to  a  six 
per  cent,  solution  of  ammonium  carbonate,  and  enough 
ammonium  phosphate  is  added  to  effect  complete  solution  ; 
then  a  delicate  white  turbidity  results  on  addition  of  zinc 
phosphate.  To  free  the  solution  of  metallic  precipitate,  it 
is  passed  through  a  hardened  filter,  is  well  washed,  and  the 
filtrate  is  saturated  with  ammonium  sulphate.  In  this  way 
a  precipitate  is  obtained  that  contains  the  diphtheria-toxin. 
A  portion  of  the  peptone  adherent  to  the  toxin  is  separated 
by  solution  in  water  and  precipitation  with  sodium  sulphate, 
but  the  albumin  can  not  be  completely  eliminated,  even  by 
repeated  washings. 

Brieger  and  Boer  have,  therefore,  cultivated  the  diph- 
theria-bacillus upon  dialyzed  urine  of  human  beings,  and 
have  prepared  from  this  albumin-free  culture-medium  by 
the  methods  just  described  a  toxin  free  from  peptone 
and  albumin.  This  toxin  is  destructive  to  animals,  and 
the  conditions  found  after  death  are  characteristic.  It 
does  not  yield  the  usual  reactions  of  albumin,  and  is  unin- 
fluenced by  reducing  substances,  but  it  is  almost  immedi- 
ately destroyed  by  oxidizing  substances.  Acids  also  de- 
stroy the  purified  toxin  of  Brieger  and  Boer,  while  feeble 
alkalies  do  not. 

By  means  of  a  concentrated  solution  of  ammonium 
chlorid  it  is  possible,  as  Brieger  and  Boer,  further,  have 
shown,  to  free  the  bodies  of  the  bacteria  completely  of  the 
specific  poison.  If  the  bacteria  thus  treated  are  ground  to 
powder,  and  small  amounts  of  this  suspended  in  water  are 
injected  into  guinea-pigs,  the  animals  die  with  necrosis  and 
suppuration  at*  the  point  of  infection.  The  bodies  of  the 
bacilli  are,  therefore,  possessed,  further,  of  a  necrotic 
poison. 


DIPHTHERIA.  213 

Relations  of  the  Bacilli  to  Diphtheria  in  Human 
Beings. — The  diphtheria-bacillus  may  be  found  in  all  cases 
of  diphtheria  in  the  diphtheric  membrane,  and  in  cases  of 
diphtheria  of  the  tonsils  throughout  the  cavity  of  the 
mouth.  It  lies  superficially,  usually  in  large  number  ;  it 
rarely  penetrates  deeply.  In  the  bodies  of  patients  suffer- 
ing from  diphtheria  the  bacillus  has  almost  never  been  found 
at  any  other  place  than  in  the  false  membrane,  and 
especially  never  in  the  blood.  Only  in  the  bodies  of 
patients  dead  of  diphtheria  has  it  been  detected  on  several 
occasions  also  in  the  blood  and  in  the  viscera.  Diphtheria 
in  human  beings  must  thus  be  viewed  as  a  foxic  infectious 
disease.  The  bacillus  is  responsible  only  for  the  local  pro- 
cess ;  the  constitutional  manifestations  (fever,  palsy,  etc.) 
being  dependent  in  human  beings  also  upon  absorption  of 
the  toxic  metabolic  products  of  the  bacillus.  The  local 
action  of  the  diphtheria-bacillus  consists  in  necrosis  of  the 
epithelium  and  of  the  uppermost  layer  of  the  mucosa, 
which  are  thus  converted  into  diphtheric  membrane.  This 
true  diphtheric  inflammatory  process  does  not  occur — apart 
from  the  action  of  certain  poisons,  as,  for  instance,  diph- 
theria of  the  large  intestine  in  conjunction  with  mercurial 
poisoning — in  the  absence  of  diphtheria-bacilli.  The  diph- 
theria-bacillus, however,  by  no  means  always  gives  rise  to 
such  a  necrotic  inflammatory  process,  when  present  in  the 
body.  The  organism  has  been  found  repeatedly  also  in 
cases  of  fibrinous  rhinitis  and  of  croupous  conjunctivitis,  the 
latter  being  clinically  quite  different  from  diphtheria  of  the 
conjunctiva.  Under  these  conditions  the  diphtheria-bacilli 
have  simply  given  rise  to  a  croupous  exudation  without 
necrosis.  Finally,  Loffler  has  found  virulent  diphtheria- 
bacilli  in  the  mouth  of  a  healthy  child,  an  observation  that 
has  since  been  repeated  by  others.  The  diphtheria-bacillus 
may,  thus,  under  certain  conditions,  vegetate  upon  mucous 
membranes  in  a  virulent  state,  without  occasioning  disease 
of  those  structures. 

Mixed  Infeption  in  Human  Beings. — The  diphtheria- 
bacillus  is  rarely  found  alone  in  the  membranes,  being 
usually  associated  with  streptococci,  also  with  staphylococci, 
pneumococci,  and  coli  commune.  It  is  almost  certain  that 
the  severe  purulent  and  septic  manifestations  observed  in 
some  cases  of  diphtheria  are  to  be  attributed  to  such  mixed 
infection  with  especially  virulent  streptococci.     It  is  a  matter 


214  CLINICAL   BACTERIOLOGY. 

of  interest  in  this  connection  that  Roux  and  Yersin  have 
shown  that  attenuated  diphtheria-bacilli  can  be  rendered 
again  virulent  by  simultaneous  inoculation  with  virulent 
streptococci. 

Saprophytic  Occurrence  of  Diphtheria-bacilli  outside 
the  human  body  has  not  yet  been  observed. 

Pseudo-diphtheria-bacilli. — Hoffmann  Wellenhof,  and 
independently  of  him  Loffler,  cultivated  as  early  as  1887  a 
microorganism  bearing  an  extraordinary  resemblance  to  the 
specific  diphtheria-bacillus,  and  which  Loffler  designated 
pseudo-diphtheria-bacillus.  Since  then,  reports  of  obser- 
vations of  this  or  quite  similar  bacteria  have  multiplied,  so 
that  a  whole  series  of  pseudo-diphtheria-bacilli  obtained 
from  cases  of  angina,  rhinitis,  from  the  healthy  mucous 
membrane  of  the  mouth,  pharynx,  and  nose,  and  from  the 
skin  and  its  diseases  is  now  known.  The  confusion  became 
still  greater  when  the  xerosis-bacilli  were  included  in  this 
group,  and  when  it  was  later  shown  that  these  bacilli  are 
present  upon  the  normal  conjunctiva. 

The  pseudo-diphtheria-bacillus  exhibits  the  same  varia- 
tions in  form  as  the  true  diphtheria-bacillus.  The  short  bacil- 
lus is  said  to  occur  more  commonly,  but  this  is  by  no  means 
always  the  case.  According  to  M.  Neisser  the  pseudo-diph- 
theria-bacilli exhibit  negative  manifestations  when  double 
staining  with  methylene-blue  acetate  and  vesuvin  is  em- 
ployed. Only  rare  examples  of  certain  varieties  of  xerosis- 
bacilli  take  the  stain.  In  applying  this  staining  reaction 
the  conditions  laid  down  (pp.  207,  208)  must  be  strictly 
observed,  as  this  means  of  differentiation  does  not  suffice  for 
cultures  several  days  old.  Further,  impression-preparations 
from  serum-cultures  six  hours  old  (p.  208),  according  to 
Neisser,  constitute  a  serviceable  aid  in  differential  diagnosis. 
The  pseudo-diphtheria-bacilli  are  not  arranged  in  a  typical 
manner,  and  after  the  lapse  of  the  short  interval  of  time  men- 
tioned they  do  not  exhibit  the  uniformly  slender,  rather  long 
form.  The  xerosis-bacilli  develop  but  slightly  in  the  course 
of  six  hours  ;  they  adhere  so  firmly  to  the  nutrient  medium 
that  typical  accumulation  does  not  appear  in  the  impression- 
preparation.  The  bacilli  exhibit  thickenings,  swellings — 
in  short,  appear  older  than  diphtheria-bacilli  cultivated  for 
six  hours  upon  serum  usually  do.  In  gelatin  stab-cultures 
the  pseudo-diphtheria-bacilli  form  small  colonies  that  spread 
upon  the  surface,  and  after  two  days  quickly  grow  larger. 


DIPHTHERIA.  215 

In  agar  streak -cultures  large,  grayish-white  colonies  form, 
that  soon  give  rise  to  extensive  elevated  deposits  ;  whereas, 
in  the  case  of  the  diphtheria-bacillus,  development  extends 
only  a  short  distance  from  the  line  of  inoculation.  On 
serum  the  colonies  soon  become  larger,  brighter,  and  softer 
than  those  of  the  diphtheria-bacillus.  In  bouillon  they 
form  a  precipitate,  clarification  taking  place  after  the  lapse 
of  three  weeks.  Upon  alkaline  potato  slight  growth  takes 
place,  and  in  milk  and  egg  a  good  growth.  Milk  is  not 
coagulated.  The  xerosis-bacilli  develop  but  slightly  upon 
all  culture-media. 

The  pseudo-diphtheria-bacilli  usually  generate  no  acid  in 
their  development  in  ordinary  bouillon  ;  individual  varieties 
form  minimal  amounts,  and  only  one  culture  of  xerosis- 
bacilli  is  mentioned  by  Neisser  that  exhibited  as  great  an 
increase  in  acidity  as  is  observed  in  the  case  of  diphtheria- 
bacilli. 

On  the  whole,  the  pseudo-diphtheria-bacilli  and  the 
xerosis-bacilli  are  not  pathogenic  for  guinea-pigs,  although, 
according  to  Spronck  and  C.  Frankel,  there  are  some 
varieties  that  exhibit  a  certain  degree  of  virulence  for  these 
animals  (causing  edema,  and  after  inoculation  of  three  cubic 
centimeters  loss  of  weight,  etc.).  The  guinea-pigs  could 
not  be  protected  against  the  action  of  these  pseudo-bacilli 
by  previous  injection  of  diphtheria-antitoxin.  The  observ- 
ers named,  therefore,  reached  the  conclusion  that  diphtheria- 
bacilli  and  pseudo-diphtheria-bacilli  are  not  identical.  Roux 
and  Yersin,  however,  take  the  position  that  the  pseudo- 
diphtheria-bacillus  represents  an  attenuated  or  temporarily 
nonvirulent  diphtheria-bacillus.  This  view  still  has  ad- 
herents, who  support  their  contention  with  the  fact  that  in 
every  case  of  true  severe  diphtheria  nonvirulent  as  well  as 
virulent  diphtheria-bacilli  are  always  observed.  The  diph- 
theria-bacillus would,  accordingly,  be  somewhat  analogous 
to  the  pneumococcus,  which,  likewise,  is  a  common  inhabi- 
tant of  the  healthy  mouth. 

The  Susceptibility  of  Human  Beings  to  Diphtheria 
can  not  be  considered  as  great,  in  view  of  the  possibility 
of  the  presence  of  diphtheria-bacilli  upon  the  mucous  mem- 
brane of  healthy  persons.  It  would  appear  that  a  special 
predisposition  on  the  part  of  the  mucous  membranes  or 
special  virulence  on  the  part  of  the  bacilli  is  necessary  for 
the  development  of  the  disease.      The  first  years  of  life  have 


216  CLINICAL  BACTERIOLOGY. 

distinctly  a  greater  susceptibility  than  adults,  but  this  grows 
less  after  the  fifth  or  sixth  year. 

The  portal  of  infection  for  the  diphtheria-bacillus 
may  be  constituted  by  any  mucous  membrane,  as  that  of 
the  nose,  the  pharynx,  and  the  larynx,  as  well  as  that  of 
the  vagina,  the  conjunctiva,  etc.,  and,  further,  every  wound- 
surface.  The  larger  number  of  cases  of  diphtheria  are  at- 
tributable to  direct  association  with  diphtheria-patients. 
The  diphtheria-bacilli  may,  under  such  conditions,  gain  en- 
trance into  the  pharynx,  where  infection  takes  place  most 
commonly ;  as  well  as  with  the  food  and  by  way  of  the  air- 
passages.  The  diphtheria-bacillus  is  probably  not  carried 
great  distances  through  the  air.  It  has  never  been  found 
in  the  air,  and  it  is  rather  sensitive  to  drying  in  a  thin  layer. 
In  thicker  layers  the  bacilli  may  survive  for  four  months, 
and  if  the  drying  be  incomplete,  for  seven  months.  Proba- 
bly the  bacilli  are  conveyed  directly  through  the  agency  of 
the  portions  of  membrane  coughed  up  by  the  patients.  In 
some  cases  eating-utensils  and  drinking-utensils,  handker- 
chiefs, toys,  etc.,  have  been  the  means  of  conveying  the 
disease-germs  from  children  suffering  from  diphtheria,  as 
well  as  convalescents.  Children  may  also  be  infected  by 
adults  through  kissing,  if  these  have  suffered  from  a  harm- 
less angina  clinically  free  from  diphtheric  characteristics.  In 
other  cases  direct  infection  can  absolutely  not  be  demon- 
strated, and  at  times  such  an  occurrence  can  be  actually 
excluded — as,  for  instance,  when  the  first  case  of  diph- 
theria occurs  in  a  village  cut  off  from  all  communication 
with  the  external  world.  Under  such  circumstances  it  must 
be  concluded  that  the  diphtheria-bacilli  have  been  derived 
from  a  case  of  diphtheria  that  pursued  the  clinical  course 
of  a  benign  angina  and  gave  rise  to  no  suspicion  of  diph- 
theria ;  or  from  a  case  already  cured,  in  which  the  bacilli 
may  be  present  for  a  long  time  after  the  disappearance  of 
all  symptoms  (p.  230)  ;  or  the  conjunction  of  special  cir- 
cumstances has,  in  accordance  with  the  theory  of  Roux  and 
Yersin,  endowed  with  virulence  pseudo-diphtheria-bacilli 
that  have  been  present  in  the  mouth  of-  the  infected  indi- 
vidual. What  conditions  may  bring  about  this  circum- 
stance, or  whether  it  is  at  all  possible,  is  not  yet  known. 
On  the  other  hand,  it  is  known,  from  an  observation  in 
which  virulent  bacilli  were  found  in  the  mouth  of  a  healthy 
child,  that  a  certain  predisposition,  a  lesion  of  the  mucous 


DIPHTHERIA.  217 

membrane,  or  the  like,  must  be  present  for  diphtheria  to 
result  from  infection  with  true  diphtheria-bacilli  from  a  case 
of  diphtheria. 

Method  and  Significance  of  the  Bacteriologic  Diag- 
nosis of  Diphtheria. — Every  case  of  inflammation  of  the 
throat  should  be  examined  with  regard  to  the  presence  of 
diphtheria-bacilli,  and,  if  these  be  found,  the  case  should  be 
treated  from  both  the  medical  and  the  hygienic  standpoint 
as  one  of  diphtheria  (just  as  every  case  of  diarrhea  with 
comma-bacilli  in  the  stools  should  be  treated  as  one  of 
cholera),  as  the  mildest  case  of  diphtheric  angina  may  give 
rise  to  severe  infection. 

For  diagnostic  purposes,  simple  microscopic  examination 
of  the  mucus  or  of  the  membrane  in  stained  cover-glass 
preparations  will  suffice  in  some  cases.  Double  staining, 
by  the  method  of  M.  Neisser,  which  is  so  characteristic  for 
preparations  made  from  cultures,  has  not  proved  entirely 
reliable  in  the  study  of  original  preparations.  If  the  prepara- 
tions, stained  single  or  double,  are  not  entirely  convincing, 
cultural  investigation  must  be  additionally  undertaken. 

A  small  portion  of  the  diphtheric  membrane  is  removed 
with  forceps,  sterilized  by  heat,  or,  directly,  with  a 
strong  platinum  loop  ;  or  a  sterile  swab  of  cotton  or  a 
sterile  bit  of  sponge  is  rubbed  upon  the  suspected  surface 
to  be  examined,  and  six  or  eight  strokes  are  made  upon  a 
plate  of  Loffler's  blood-serum.  Should  this  not  be  avail- 
able, from  three  to  five  tubes  of  blood-serum  or  glycerin- 
agar,  solidified  in  slants,  are  successively  inoculated.  Plates 
and  tubes  are  placed  in  the  thermostat  at  a  temperature  of 
37°  C.  (98.6°  F.) — according  to  M.  Neisser  best  at  a 
temperature  of  34°  C.  (93.2°  F.)  or  35°  C.  (95°  F.). 
In  the  first  streak  or  in  the  first  tube  inoculated  the 
colonies  are  too  dense,  and  they  coalesce  ;  in  the  last  they 
are  isolated,  and  these  are  then  examined  further.  It  is 
recommended  also  that  the  membranes,  before  being 
smeared,  be  rinsed  for  several  minutes  in  two  per  cent,  solu- 
tion of  boric  acid,  whereby  a  considerable  number  of  the 
saprophytic  bacteria,  accidentally  present,  are  removed,  so 
that  a  separation  of  the  individual  colonies  is  brought  about 
in  the  first  inoculation-smears.  After  six  or  eight  hours 
impression-preparations  are  made  from  the  serum-plate. 
If  these  display  the  typical  collections  described  (p.  208), 
then  a  positive  diagnosis  can  be  made.     A  negative  diag- 


218  CLINICAL  BACTERIOLOGY. 

nosis,  however,  should  be  made  only  with  great  reserve 
after  the  lapse  of  only  six  hours  (M.  Neisser).  After 
eighteen  or  twenty  hours  smear-preparations  are  made,  and 
these  are  treated  by  the  method  of  double  staining  already 
described  (p.  207).  Typical  granules  in  typical  bacilli  may 
be  considered  as  conclusive,  although  Neisser  himself  issues 
a  warning  against  dependence  upon  double  staining  alone 
until  his  observations  have  been  amply  confirmed  by  others. 
If,  in  accordance  with  older  methods,  dependence  is  placed 
upon  simple  staining  of  an  ordinary  cover-slip  preparation 
from  a  plate  or  a  test-tube  of  Loffler's  serum,  the  possi- 
bility of  confusion  with  pseudo-diphtheria-bacilli  must  con- 
stantly be  borne  in  mind.  It  is  then ,  necessary  to  scrutinize 
carefully  the  peculiarities  that  have  been  described  as  in- 
dicating either  diphtheria  or  pseudo-diphtheria.  No  single 
feature  should  be  accepted  as  conclusive  in  the  diagnosis  ; 
but  the  conjunction  of  all  should  be  required.  Frequently, 
experiments  on  animals  must  be  resorted  to  in  order  to 
determine  whether  the  bacilli  are  virulent  or  not.  If  they 
prove  virulent,  the  organisms  are  probably  without  question 
true  diphtheria-bacilli.* 

Immunity  and  Specific  Therapy. — Diphtheria  may 
attack  the  same  child  on  several  occasions.  It  is  not  one 
of  those  diseases  that  leave  behind  them  permanent  immu- 
nity, but  rather  one  of  those  that  predispose  to  recurrence. 
Nevertheless,  it  is  certain  that  after  recoveiy  from  diph- 
theria some  degree  of  immunity  exists  temporarily.  This 
is  demonstrated  by  the  observations  of  Escherich,  Kle- 
mensiewicz,  and  others,  who  showed  that  the  blood- 
serum  of  children  convalescent  from  diphtheria  exhibits 
immunizing  properties. 

Experimental  animals  are  readily  immunized  to  diph- 
theria. Behring  and,  later,  Roux  conferred  such  immunity 
on  a  large  scale  upon  horses  by  preliminary  treatment  with 
diphtheria-toxin  whose  toxicity  was  attenuated  by  addition 
of  iodin  trichlorid  or  solution  of  iodin  and  potassium  iodid. 
By  the  introduction  of  gradually  increasing  amounts  of 
diphtheria-toxin  the  immunity  of  these  animals  was  in- 
creased to  a  high  degree. 

*  The  method  commonly  practised  in  public  laboratories  consists  in  inocula- 
tion of  a  tube  of  Loffler'  s  serum  with  a  previously  sterilized  swab  applied  to  the 
suspicious  membrane,  cultivation  in  the  incubator  overnight,  and  microscopic 
examination  of  cover-slip  preparations  stained  with  Loffler' s  solution. — A.  A.  E. 


DIPHTHERIA.  219 

The  following  instance,  taken  from  the  text-book  of 
Mace,  will  illustrate  the  immunization  of  a  horse  by  the 
method  of  Roux.  For  the  better  comprehension  of  the 
description  it  should  be  mentioned,  as  has  already  been 
pointed  out  in  the  consideration  of  the  physiology  of  the 
disease  (p.  211 ),  that  the  toxin  is  the  equivalent  of  the  fil- 
trate of  a  bouillon-culture,  or  of  a  culture  in  which  the 
bacilli  have  been  destroyed  by  addition  of  carbolic  acid  in 
a  proportion  of  o.  5  per  cent.  : 

Toxin  with  Addi- 
Day.  Injection        tion  of  Iodin- 

OF  potassium  Iodid. 

I j^  cu.  cm.  I  :  10  No  reaction. 

2 /^  cu.  cm.  I  :  10  '* 

4,  6,  8     ....  ^  cu,  cm.  I  :  10  *' 

13,  14 I  cu.  cm.  I  :  10  " 

17 J4^  cu.  cm.  Pure  toxin  Slight  edema,  without  fever. 

22 I  cu.  cm.  *'  '*                       " 

23 2  cu.  cm.  "  **                       ** 

25 3  cu.  cm.  *<  «*                        *« 

28 5  cu.  cm.  *'  *<                        <* 

30,  32,  36   .    .    .  5  cu.  cm.  '<  «                        ** 

39,  41      ...    .  10  cu.  cm.  **  "                        *' 

43,  46,  48,  50    .  30  cu.  cm.  "  Marked  edema,  disappearing 

in  the  course   of  twenty- 
four  hours. 

53 60  cu.  cm.  "  **                       ** 

57>  63,  65,  67    .  60  cu.  cm.  *<  **                      ** 

72 90  cu.  cm.  **  "                       ** 

80 250  cu.  cm.  '*  "                       " 

Small  doses  of  pure  toxin  may  also  be  employed  in  the 
first  inoculations.  Some  horses  bear  from  the  beginning 
I  cu.  cm.  of  a  toxin  of  which  ^  cu.  cm.  is  sufficient  to 
destroy  within  forty-eight  hours  a  guinea-pig  weighing 
300  grams.  The  immunity  can  be  looked  upon  as  well 
established  after  the  animal  has  received  from  60  to  70  cu. 
cm.  of  this  toxin.  It  is  then  possible  to  inject  much  larger 
amounts  without  injury.  In  order  to  obtain  diphtheria- 
antitoxin  it  is  best  to  employ  horses,  in  the  first  place  be- 
cause they  are  very  readily  immunized,  and  in  the  second 
place  because  they  can  be  bled  innumerable  times. 

In  immunizing  the  animals  a  normal  toxin-solution  must 
be  available  to  begin  with,  and  which  affords  a  standard  of 
comparison.  Behring  designates  as  a  diphtheida  normal 
toxin  that  diphtheria-solution  of  which  i  cu.  cm.  represents 
the  minimal  lethal  dose  for  100  guinea-pigs  each  weighing 
250  grams.     To  indicate  this  normal  toxin,  he  has   intro- 


220  CLINICAL  BACTERIOLOGY. 

duced  the  abbreviation  DTN^,  and  he  designates  a  toxin  of 
10  times  this  strength  as  DTN^^  and  one  o(  ^  this 
strength  as  -^^.  As  guinea-pigs  weighing  250  grams  are 
not  always  obtainable,  the  important  minimal  lethal  dose  is 
not  estimated  with  regard  to  the  entire  experimental  ani- 
mal, but  upon  the  basis  of  each  gram  of  living  bodily 
weight.  In  order  to  express  this  relation  also  in  simple 
symbols  Behring  designates  the  whole  animal  with  an  m,* 
and  a  gram  of  the  living  bodily  weight  with  an  M.  The 
weight  of  the  guinea-pig  in  each  individual  instance  is 
added  besides,  and  above  the  letter  m  (thus,  for  instance, 
m^so  stands  for  an  animal  weighing  280  grams).  The 
minimal  lethal  dose  for  i  gram  of  body-weight — thus,  for 
I  M — is  indicated  by  prefixing  the  symbol  -{-.  +1500M 
expresses  that  amount  of  toxin  that  just  suffices  to  destroy 
6  guinea-pigs  each  weighing  250  grams.  One  cubic  centi- 
meter of  DTN^  causes  the  death  of  100  m^^o^  ^nd  is  thus 
equal  to  +25,000  M — that  is,  it  represents  25,000  minimal 
lethal  doses  for  each  gram  of  living  guinea-pig  by  weight. 
Of  diphtheria-toxin  10  times  the  normal  strength,  DTN^^, 
I  cu.  cm.  =  +  250,000  M;  i  cu.  cm.  of  diphtheria-toxin 
one-tenth  the  normal  strength,  -^^,  ^+  2500  M. 

In  order  to  obtain  an  active  diphtheria-toxin  it  is  ob- 
viously necessary  to  employ  highly  virulent  diphtheria- 
bacilli  in  preparing  the  bouillon-cultures.  As  a  consequence 
it  is  frequently  necessary  to  fortify  the  virulence  of  the 
diphtheria-bacilli.  A  diphtheria-bacillus  that  has  com- 
pletely lost  its  property  of  generating  toxin  can  not  be 
rendered  virulent  again  by  any  method  thus  far  known. 
If,  however,  a  trace  of  virulence  remain,  this  may  be 
augmented  by  repeated  passage  through  animals.  Six  or 
eight  hours  after  the  injection  a  specimen  is  obtained  from 
the  area  of  local  edema,  and  with  it  a  culture  is  made,  which 
then  serves  for  the  inoculation  of  a  new  animal.  Roux 
and  Yersin  augmented  and  accelerated  the  production  of 
toxin  on  the  part  of  the  diphtheria-bacilli  by  cultivating  these 
in  a  shallow  layer  of  bouillon  through  which  a  current  of 
moist  air  was  constantly  passed.  The  diphtheria-bacillus 
thrives  vigorously  in  nutrient  media  containing  sugar, 
although  it  generates  considerably  less  toxin. 

■^In  the  original,  the  German  capital  letter  9Ji  is  employed,  but  for  conve- 
nience sake  the  English  small  letter  m  is  here  substituted. — A.  A.  E. 


DIPHTHERIA.  221 

♦ 

When  the  horses  have  been  rendered  sufficiently  immune, 
five  or  six  liters  of  blood  are  withdrawn  from  the  jugular 
vein  by  means  of  a  trocar,  eight  or  ten  days  after  the  last 
injection.  The  blood,  after  standing  in  the  refrigerator, 
yields  a  clear  serum,  which  exhibits  its  maximum  activity 
eight  or  ten  days  after  the  injection  of  toxin.  If  the  blood 
is  removed  earlier,  the  serum  is  considerably  weaker. 
After  the  period  of  maximum  activity,  which  persists  for 
several  days,  a  gradual  reduction  takes  place,  which  may 
lead  to  complete  disappearance  of  the  antitoxin,  unless 
meanwhile  new  injections  of  toxin  are  made.  For  purposes 
of  preservation  carbolic  acid,  in  the  proportion  of  0.5  per 
cent,  is  added  to  the  antitoxin  (Behring,  Hochst  serum),  or 
0.4  per  cent,  trikresol  (Aronsohn,  Schering  serum),  or  a  bit 
of  camphor  (Pasteur  Institute). 

It  has  been  pointed  out  in  the  general  section  that 
toxin  and  antitoxin,  admixed  in  a  test-tube,  mutually 
neutralize  the  activity  of  each  other,  so  that  when  injected 
simultaneously  into  guinea-pigs,  no  manifestations  of  disease 
follow.  In  this  mutual  interaction  a  regular  gradation  is  ob- 
servable. In  estimating  the  immunizing  value  of  the  cura- 
tive serum  Behring  and.Ehrlich  did  not  proceed  from  the 
simple  lethal  dose,  but  from  ten  times  that  amount.  They 
designate  as  normal  serum  that  of  which  o.  i  cu.  cm. 
suffices  to  neutralize  ten  times  the  lethal  toxic  dose,  so  that 
a  guinea-pig  weighing  250  grams  withstands  the  injection 
without  injury.  One  cubic  centimeter  of  this  serum  repre- 
sents a  normal  antitoxin-unit.  The  further  calculation  can  be 
readily  made  upon  this  basis.  A  serum,  for  instance,  of 
which  0.0 1  cu.  cm.  neutralizes  10  times  the  lethal  dose 
represents  10  times  the  strength  of  normal  serum — that  is, 
I  cu.  cm.  contains  10  normal  antitoxin-units.  Should 
o.ooi  cu.  cm.  of  a  serum  suffice  for  this  purpose,  it  repre- 
sents a  strength  100  times  that  of  normal  serum,  and  i 
cu.  cm.  contains  100  normal  antitoxin-units. 

Behring  subsequently  introduced  a  new  method  of  cal- 
culation. He  designates  as  diphtheria  normal  antitoxin, 
DANi,  that  serum  of  which  i  cu.  cm.  neutralizes  i  cu.  cm. 
of  diphtheria  normal  toxin,  DTN^,  =  -|-  25,000  M.  These 
figures  he  converts  directly  into  weight  of  guinea-pigs,  in 
grams,  M,  by  simply  prefixing  the  minus  sign.  DAN^ 
thus  equals  —25,000  M  :  that  is,  i  cu.  cm.  of  diphtheria 
normal  antitoxin  is  capable  of  neutralizing  the  lethal  dose 


222  CLINICAL   BACTERIOLOGY. 

for  25,000  grams  in  weight  of  guinea-pig.  As  may  be 
readily  seen,  this  mode  of  calculation  agrees  entirely  with 
the  first  described.  According  to  that,  o.  i  cu.  cm.  of 
serum  was  capable  of  neutralizing  10  times  the  minimal 
lethal  dose  ;  while  according  to  this,  i  cu.  cm.  of  serum  is 
capable  of  neutralizing  100  times  the  minimal  lethal  dose  in 
the  typical  guinea-pig  weighing  250  grams. 

Roux  estimates  the  immunizing  power  in  quite  another 
manner.  He  determines  how  much  serum  must  be  injected 
from  twelve  to  twenty-four  hours  previously,  in  order  to 
protect  a  guinea-pig  against  the  lethal  dose  of  diphtheria- 
bacilli  or  of  diphtheria-toxin  that  otherwise  would  have  de- 
stroyed the  animal  in  not  more  than  thirty  hours.  He  fixes 
as  the  power  of  the  serum  that  figure  which  expresses  the 
relation  between  the  amount  of  serum  and  the  body-weight 
of  the  guinea-pig.  An  immunizing  power  of  20,000  in- 
dicates thus  that  of  this  serum  -2-fo"o"o  P^^^  ^^  ^^^  body- 
weight  of  a  guinea-pig  must  be  injected  in  order  to  protect 
the  animal  against  the  amount  of  diphtheria-culture  or 
diphtheria-toxin  that  would  cause  death  in  thirty  hours. 

Spronck  has  proposed  a  special  formula  for  converting 
the  immunity-unit  of  Roux  into  that  of  Behring.  B  =  ^ 
— that  is,  it  is  only  necessary  to  divide  Roux's  figures  by 
500  in  order  to  obtain  the  number  of  Behring  units. 

In  a  communication  upon  the  estimation  of  the  strength 
of  diphtheria-antitoxin  and  its  theoretic  basis  Ehrlich  starts 
with  a  dry  diphtheria-antitoxin  prepared  in  the  Hochst 
works  in  order  to  obtain  a  sufficiently  constant  standard  and 
a  unit  of  measure  for  the  serum  that  is  protected  from  the 
destructive  influence  of  water,  oxygen,  light,  and  heat. 
This  contains  1 700  immunization-units  or  normal  antitoxin- 
units  to  the  gram.  It  is  preserved  in  a  small  apparatus  con- 
sisting of  two  communicating  glass  tubes,  of  which  one  con- 
tains the  powder  and  the  other  phosphoric  anhydrid  as  a 
dehydrating  agent.  The  apparatus  is  most  carefully  ex- 
hausted of  air,  and  kept  in  a  dark,  cool  place.  Each  tube 
contains  2  grams  of  antitoxin-powder,  which  before  being 
used  is  dissolved  in  200  cu.  cm.  of  a  solution  of  sodium 
chlorid  (10  per  cent.)  and  glycerin  (from  50  to  80  per 
cent).  One  cubic  centimeter  of  this  solution  diluted  17 
times  represents  the  immunity-unit  or  normal  antitoxin-unit. 
If  to  this  immunity-unit  are  added  increasing  quantities  of 
diphtheria-toxin,  it  is  possible  to  fix  two  limits  (L),  which 


DIPHTHERIA.  223 

are  of  great  importance  for  the  characterization  of  the 
toxin.  The  first  (Lq)  represents  that  amount  of  toxin  that 
is  neutraHzed  by  the  serum,  so  that  injection  of  the  mixture 
is  borne  by  the  guinea-pig  without  injury.  The  second 
hmit  (L  ^)  represents  the  amount  on  injection  of  which  such 
a  marked  excess  of  toxin  is  rendered  active  in  spite  of  the 
presence  of  the  anti-body  that  death  of  the  guinea-pig  takes 
place  within  four  days.  According  to  the  foregoing  con- 
siderations, Lq  represents  about  lOO  lethal  doses  ;  for,  as 
has  been  explained,  if  the  antitoxin-unit  DAN  ^  neutralizes 
the  toxin-unit  DTN  i,  that  is  i  cu.  cm.  of  that  toxin  of 
which  o.oi  cu.  cm.  destroys  a  guinea-pig  weighing  250 
grams  ;  and  the  difference  (D)  between  the  two  limits  (L^ 
and  L  J  should  be  equal  to  the  minimal  lethal  dose.  These 
assumptions  are,  however,  not  justified,  as  Ehrlich  has  shown 
as  the  result  of  most  painstaking  experiment.  L  ^  varies  be- 
tween 27  and  109  toxin-doses,  and  D  between  i,  7,  and  28. 
Ehrlich,  upon  the  basis  of  experiments  with  tetanus,  had 
already  come  to  the  conclusion  that  by  the  action  of  carbon 
disulphid  tetanus-toxin  could  be  transformed  into  an  in- 
nocuous modification,  but  which  is  still  capable,  both  in  the 
test-tube  and  in  the  animal  body,  of  combining  with  the 
anti-bodies.  The  spontaneous  attenuation  of  the  diphtheria- 
toxin  so  frequently  encountered,  without  the  slightest  dimi- 
nution in  its  neutralizing  activity,  is  attributed  by  Ehrlich 
to  a  similar  transformation  of  a  portion  of  the  toxin  into 
such  toxin- modifications,  and  for  these  he  proposes  the 
name  of  toxoids. 

Ehrlich  divides  the  toxoids  into  three  groups  :  (i)  Pro- 
toxoids,  which  unite  with  the  antitoxin  more  easily  than 
the  toxin  ;  (2)  syntoxoids,  which  exhibit  for  the  antitoxin 
the  same  affinity  as  the  toxin ;  and  (3)  epitoxoids,  which 
possess  a  lesser  attraction  for  the  antitoxin.  As  the  pro- 
toxoids  possess  a  greater  affinity  for  the  anti-bodies  than,  and 
the  syntoxoids  the  same  affinity  as,  the  toxin,  they  can  not 
be  displaced  by  the  toxin  from  their  combination  with  the 
anti-toxin.  If,  therefore,  the  neutral  toxin-antitoxin -mixture 
(Lo)  consists  of  toxins,  protoxoids,  and  syntoxoids,  then 
L  ^  is  produced  by  the  addition  of  the  simple  lethal  dose, 
or,  what  amounts  to  the  same  thing,  D,  the  difference  be- 
tween Lq  and  L  ^.  is  equal  to  this  lethal  dose.  The  condi- 
tions are,  however,  quite  different  when  epitoxoids  are 
present.     These  must,  in  the  first  place,  be  displaced  by  the 


224  CLINICAL  BACTERIOLOGY. 

toxin  added,  and  then  there  must  be  present,  besides,  a  sim- 
ple toxin-unit  in  order  to  attain  L  ^  and  to  cause  the  death 
of  the  animal.  D  corresponds  here  to  the  epitoxoid-units 
plus  I  toxin-unit.  Every  normal  toxin-bouillon  contains 
the  three  toxoids.  Their  relations  may  be  represented  as 
follows  :  Diphtheria-bouillon  =  x-toxoids  (protoxoids  and 
syntoxoids)  -j-  y-toxin  -f  z-epitoxoid.  Lq,  then,  =  x- 
toxoid  saturated  -|-  y-toxin  saturated  +  z-epitoxoid  satu- 
rated. To  determine  the  equivalent  of  «  in  toxin,  it  is  only 
necessary  to  know  the  number  of  lethal  doses  present  in 
Lq.  L+  then  corresponds  with  the  formula :  x-toxoid 
saturated  -|-  (y  +  z)  toxin  saturated  +  1  toxin  free  -f-  z-epi- 
toxoid free.  The  number  of  epitoxoids  /?  equals,  as  has 
been  seen,  D  —  1,  but  only  in  the  event  of  the  bouillon 
being  constituted  exclusively  of  toxin  and  epitoxoid.  If 
additional  toxoids  are  present,  then  /5  expresses  a  relative 
value,  and  is,  therefore,  designated  by  Ehrlich  as  a  function 
of  /5,  thus,  F  (/?).  The  formula  for  the  toxin-bouillon  may, 
therefore,  be  expressed  as  follows  :  x-toxoids  -j-  « toxin  -j- 
F  (/5)  epitoxoid. 

For  the  majority  of  diphtheria-poisons  it  may  be  shown 
by  addition  of  the  simple  immunity-unit  that  they  originally 
contained  the  required  lOO  doses  of  toxin.  The  attenua- 
tion is  effected  gradually  according  to  the  principle  of  thirds 
or  halves.  Of  three  toxin-molecules  two  are  converted 
into  toxoids,  or  1  toxin  is  converted  into  equal  parts  of 
toxoids  and  toxin.  It  appears  that  in  this  process  of  de- 
composition in  the  cold  no  epitoxoid — which  always  occurs 
in  cultures  maintained  at  a  temperature  of  37°  C.  (98.6°  F.) 
— is  formed,  but  only  protoxoids  and  syntoxoids.  In  the 
study  of  an  especially  active  toxin  immediately  after  its 
acquisition  Ehrlich  obtained  the  following  figures  :  L+  = 
100  doses  of  toxin  ;  L^  =  50  doses  of  toxin.  The  toxin, 
thus,  was  of  half  strength,  and  the  figures  obtained  had, 
therefore,  to  be  multiplied  by  2.  The  value  of  L^.,  then, 
equaled  200,  and  the  formula  for  the  bouillon  was  as 
follows  :  50  toxoids  +  50  toxin  +  100  epitoxoids.  As  a  re- 
sult of  this  demonstration  that  the  antitoxin-unit  saturates 
200  toxin-equivalents,  that  the  poison  itself  is  attenuated 
dichotomously,  it  was  possible  without  difficulty  to  explain 
the  previously  mysterious  manifestation  that  with  freshly 
prepared  toxins  frequently  just  100  toxin-equivalents  are 
neutralized  by  the  immunity-unit. 


DIPHTHERIA.  225 

The  total  of  toxoids,  toxins,  and  epitoxoids,  equals, 
according  to  Ehrlich,  200 ;  a  toxin  possessing  the  a 
equivalent  of  toxin  and  z-epitoxoid  should  yield  the  following 
formula:  (200  — a  —  z)  toxoids  -f  «  toxin  +  z-epitoxoids. 
To  this  the  immunity-unit  is  added,  and  in  this  way  is  ob- 
tained the  value  Lq  =  (200-a-z)  toxoid-antitoxin  -f  a 
toxin-antitoxin  +  z-epitoxoid-antitoxin.  The  value  of  L_|. 
is  obtained  by  adding  to  the  neutral  mixture  so  much 
of  the  original  material  that  the  z-epitoxoid-antitoxin  is 
decomposed  by  the  mixture  of  toxoid  -|-  toxin.  As  is 
evident,  this  addition  must  represent  a. ^^^  toxin-units.  The 
epitoxoid  toxin-units  have  just  been  found  to  be  /9  =  D  —  i. 
It  results  that  /5  ^  «•— J—  and  z  =  -^^.      If  the  amount  of 

200 — z  a  +  P 

epitoxoid  is  estimated  according  to  this  formula,  figures  are 
obtained  that  stand  in  the  simplest  relation  to  that  found 
for  the  number  of  immunity-units,  200 — namely,  ^,  y^y 
;^,  or  i^,  or  ^  thereof 

As  the  outcome  of  these  experiences,  Ehrlich  has  sug- 
gested the  following  alterations  in  the  directions  for  testing 
the  diphtheria-antitoxin,  and  these  have  been  confirmed 
by  a  decree  dated  March  29,  1 897  : 

I.  As  a  standard  for  the  estimation  of  the  antitoxin  an 
antitoxin-powder  of  accurately  determined  strength,  pro- 
tected against  the  influence  of  oxygen  and  water,  is  em- 
ployed. This  is  contained  in  carefully  measured  quantities 
in  specially  prepared  vacuum-tubes.  The  apparatus  at 
the  time  present  in  the  laboratory  are  filled  each  with  2 
grams  of  a  dry  antitoxin  1700  times  the  normal  strength. 

II.  To  secure  the  greatest  possible  degree  of  permanence 
the  antitoxin  should  be  dissolved  in  a  mixture  of  equal 
parts  of  10  per  cent,  solution  of  sodium  chlorid  and 
glycerin.  A  tube  is  to  be  opened  every  three  months  and 
a  new  solution  prepared.  Of  the  dry  antitoxin  at  the  time 
preserved  in  the  laboratory  the  contents  of  a  tube  are 
dissolved  in  200  cu.  cm.  of  the  mixture  described,  and  thus 
a  test  antitoxin-solution  17  times  the  normal  strength  is 
prepared. 

III.  The  present  test-dose  of  toxin  is  determined  with 
the  aid  of  an  immunity-unit,  such  as  is  contained,  for  in- 
stance, in  I  cu.  cm.  of  a  y^  dilution  of  the  test-antitoxin 
17  times  the  normal  strength.  To  this  amount  of  antitoxin 
increasing  amounts  of  toxin  are  added,  and  by  means  of 

15 


226  CLINICAL  BACTERIOLOGY. 

most  careful  experimental  observations  the  limit  is  deter- 
mined at  which  just  that  excess  of  toxin  becomes  manifest 
which  causes  death  of  the  animal  in  the  first  four  days. 
The  amount  of  toxin  thus  obtained  represents  the  imme- 
diate test-dose.  By  means  of  the  same  dose  of  serum,  for 
the  more  exact  characterization  of  the  toxin,  the  determi- 
nation of  a  second  limit  is  made,  for  the  purpose  of  learning 
the  dose  of  toxin  that  is  just  neutralized  by  admixture  with 
the  amount  of  serum  named. 

IV.  The  determination  of  the  strength  of  a  diphtheria- 
antitoxin  is  made  by  means  of  the  test-dose  of  toxin  (see 
paragraph  III)  as  follows:  The  test-dose  of  toxin  in  question 
— for  instance,  0.355  ^^-  cm.  of  tested  toxin  at  the  time 
present  in  the  laboratory — is  mixed  with  4  cu.  cm.  of  anti- 
toxin corresponding  to  the  test-figures  given.  As  the  test- 
dose  of  toxin  is  estimated  for  i  cu.  cm.  of  antitoxin  of 
normal  strength,  or  for  4  cu.  cm.  of  antitoxin  }^  the  normal 
strength,  an  antitoxin  of  x-strength  will  have  to  be  diluted 
i^  x,  and  in  testing  an  antitoxin  100  times  the  normal 
strength,  -^-^. 

V.  The  mixture  obtained  is  injected  unmodified  sub- 
cutaneously'  into  guinea-pigs  weighing  from  250  to  300 
grams.  If  the  animals  die  in  the  test-experiments  made  by 
two  observers  in  the  laboratory  within  the  first  four  days, 
the  antitoxin  does  not  possess  the  required  strength. 
Should  death  occur  within  five  or  six  days,  the  antitoxin 
is  close  to  the  required  strength,  and  in  order  to  avoid  the 
early  withdrawal  to  be  anticipated  an  improvement  of  from 
5  to  10  per  cent,  is  recommended  the  manufacturers.  In- 
durations that  occur  in  the  animals  experimented  upon  do 
not,  however,  constitute  sufficient  ground  for  objection. 
In  the  case  of  the  dead  animals  an  autopsy  should  be  held, 
and  careful  attention  directed  to  complications  with  previ- 
ously existing  disease  (tuberculosis,  pseudo-tuberculosis, 
and  pneumonia)  that  may  induce  undue  susceptibility  on 
the  part  of  the  test-animals. 

VI.  Both  liquid  and  solid  toxins  may  be  employed  for 
test-purposes,  if  the  limits  defined  in  paragraph  III  can  be 
accurately  estimated,  and  the  difference  between  them  does 
not  exceed  fifteen  simple  lethal  doses.  If  liquid  toxins 
preserved  in  toluol  are  employed,  this  should  be  done 
only  if  as  a  result  of  preliminary  investigation  the  perma- 
nency of  the  test-constants  is  demonstrated,   if  the  test- 


DIPHTHERIA.  227 

dose  does  not  exceed  i  cu.  cm.  The  examination  with  re- 
gard to  the  quaUties  of  the  test-toxins  should  be  continued. 
VII.  The  test-toxins,  if  hquid,  are  to  be  examined 
monthly  with  regard  to  their  sterility  by  means  of  culture- 
methods. 

VIII.  The  test-poison  is  to  be  redetermined  at  intervals 
of  six  weeks  by  means  of  the  test-dose  of  serum,  the  test- 
dose  and  the  net-valuation  being  estimated  anew.  If,  on 
reexamination,  any  considerable  deviation  in  the  test-dose 
should  be  detected,  the  toxin  must  be  considered  to  be  in 
process  of  decomposition,  and  it  should  be  replaced  by  fresh 
toxin. 

IX.  The  manufacturers  are  to  be  informed  that  the 
test-toxin  in  small  amounts  decomposes  readily,  and  that 
even  brief  exposure  to  light  may  induce  considerable  at- 
tenuation. It  is  therefore  to  be  recommended  that  a  new 
supply  of  toxin  be  obtained  from  the  laboratory  ever>-  three 
weeks.  * 

Diphtheria-antitoxin  is  prepared  in  Germany  in  four 
establishments — namely,  the  Hochst  Works,  the  Factory 
of  Schering,  in -Berlin,  the  Pasteur  Institute  in  Stuttgart,  and 
the  Factory  of  Sthamer,  Noack  &  Company  in  Hamburg. 

The  Hochst  Works  manufacture  the  following  prepara- 
tions : 

ANTITOXIN  OF  250  TIMES  THE   NORMAL  STRENGTH. 
Number  o,  yellow,  .    .0.8  cu.  cm.  contain    200  immunity-units. 
Number  i,  green,    .    .  2.4       "  "        600  " 

Number  2,  white,    .    .  4  "  "     1,000  *' 

Number  3,  red,    ...  6  "  "     1,500  *' 

*  Dr.  Jos.  McFarland  and  Dr.  Chas.  T.  McClintock,  to  whom  these  regula- 
tions were  submitted,  kindly  describe,  as  follows,  the  method  of  testing  pur- 
sued by  the  largest  manufacturers  of  diphtheria-antitoxin  in  the  United  States  : 

"  In  order  to  secure  uniformity  in  the  toxin  the  same  culture  of  the  diph- 
theria-bacillus is  always  employed.  This  is  grown  for  seven  days  at  37°  C. 
(98.6°  F.)  in  an  accurately  prepared  alkaline  two  per  cent,  peptone-bouillon. 
The  same  degree  of  alkalinity  is  always  secured  (phenolphthalein  being  used  as 
the  indicator  in  titration).  After  a.ddition  of  0.4  per  cent,  trikresol,  this 
toxic  bouillon  is  filtered  through  unglazed  porcelain  and  stored  in  a  dark,  cold 
place.  The  amount  of  this  toxin  that  will  kill  a  guinea-pig  weighing  250 
grams  on  or  before  the  sixth  day  is  considered  the  minimum  fatal  dose. 
Should  this  toxin  deteriorate  ten  per  cent,  from  its  original  strength,  it  must  be 
discarded. 

'*  In  testing  antitoxin,  a  series  of  guinea-pigs,  weighing  from  240  to  270 
grams,  are  injected  with  ten  times  the  minimum  fatal  dose  of  toxin  previously 
mixed  with  varying  amounts  of  antitoxin.  Those  pigs  are  considered  protected 
that  do  not  die  or  lose  more  than  20  per  cent,  of  their  original  weight  in  seven 
days."— A.  A.  E. 


228  CLINICAL  BACTERIOLOGY. 


STRONG  ANTITOXIN  OF  500  TIMES  THE  NORMAL  STRENGTH. 

Number  o  D,  yellow,  .  i  cu.  cm.  contains     500  immunity-units. 

Number  2D,  white,  .  2       "        contain  1,000  " 

Number  3  D,  red,     •  .3       "  "        i,5oo  " 

Number  4 D,  violet,  .  4       '*  '*        2,000  ** 

Number  6  D,  blue,    .  .  6       *'  "        3, 000  " 

ANTITOXIN  OF  600  TIMES  THE   NORMAL  STRENGTH. 
Number  6  e,  blue,     .    .  5  cu.  cm.  contain  3000  immunity- units. 

The  Pasteur  Institute,  of  Stuttgart,  manufactures  only 
one  preparation  of  50,000  Roux  immunity-units. 

The  Schering  factory  produces  the  following  antitoxins  : 

A,  100  immunity-units  per  cu.  cm. 

B,  200  *'  '*       '*         (in  vials  of  5  and  lo  cu.  cm.). 
Strong  antitoxin  of  500  times  the  normal  strength,  500  immunity- 
units  per  cu.  cm.  (in  vials  of  2  and  4  cu.  cm.). 

The  Hamburg  establishment  dispenses  diphtheria-anti- 
toxin prepared  by  Ruete  and  Enoch  and  containing  300 
immunity-units  per  cu.  cm. 

In  injecting  the  serum  the  rubber-ball  syringe  of  Koch 
may  be  employed,  and  this  is  easily  manipulated  by  one 
who  is  experienced.  (Fig.  36,  2.)  The  antitoxin-syringe  of 
Roux  is  to  be  recommended  as  especially  practical,  and 
it  can  be  sterilized  with  the  greatest  ease.  (Fig.  36,  i.)  It 
is  either  boiled  in  a  one  per  cent,  solution  of  soda,  or  it  is 
cleansed  with  a  five  per  cent,  solution  of  carbolic  acid,  after 
which  it  is  rinsed  with  a  o.  5  per  cent,  solution  in  order  that 
the  antitoxin  may  not  be  injured  in  consequence  of  too 
great  concentration  of  the  antiseptic. 

The  number  of  immunity-units  to  be  injected  in  the 
individual  case  will  depend  entirely  upon  its  severity,  and 
upon  the  day  of  the  disease  that  the  patient  comes  under 
treatment.  In  a  mild  case,  coming  under  observation  on 
the  first  day,  600  immunity-units  will  suffice.  Severe 
cases,  seen  as  late  as  the  third  day,  will  require  1000  im- 
munity-units. In  still  more  severe  cases,  seen  at  a  late 
stage,  1500  immunity-units  and  more  are  employed. 

The  results  that  have  thus  far  been  obtained  in  the 
treatment  of  diphtheria  with  the  antitoxin  have  been  ex- 
ceedingly favorable.  The  mortality  from  diphtheria  has 
been  reduced  about  half  under  the  influence  of  the  antitoxin. 
In  support  of  this  statement  we  may  quote  the  results  of 


DIPHTHERIA.  229 

the  collective  investigation  with  regard  to  the  diphtheria- 
antitoxin  for  the  period  from  April,  1895,  to  March, 
1896,  conducted  by  Dieudonne  in  the  Imperial  Health- 
office.  There  were  treated  with  antitoxin  in  hospitals 
9581  cases,  among  which  death  occurred  in  1589 — 15.5  per 
cent.  If  the  cases  still  under  treatment  at  the  conclusion 
of  the  report  be  excluded,  the  proportion  of  deaths  to 
recoveries  was  3  to  16.  In  the  eleven  years  preceding  the 
introduction  of  the  antitoxin  (from  1883  to  1893)  the  pro- 
portion of  deaths  to  recoveries  averaged  6  to  16.  Nearly 
one-half  of  the  cases  included  in  this  study  were  designated 
in  the  reports  as  severe.  The  mortality  among  children 
under  two  years  old  (11 89)  equaled  39.1  per  cent,  and 
that  of  those  subjected  to  tracheotomy  (2744)  32.3  per 
cent.* 

In  the  cases  submitted  to  treatment  with  antitoxin  the 
disease  pursued,  in  general,  a  milder  and  more  favorable 
course.  Existing  manifestations  of  stenosis  improved  in  a 
large  number  of  cases,  so  that  tracheotomy  was  avoided. 
Serious  sequelae  with  certainty  attributable  to  the  antitoxin 
have  thus  far  not  been  observed.  In  rare  cases  unpleas- 
ant complications  of  a  transient  character  occur — viz.,  in- 
filtration at  the  point  of  injection,  pains  in  the  joints  and  ex- 
tremities, urticaria,  exanthemata,  possibly  also  albuminuria. 
These  symptoms  are  not  at  all  dependent  upon  the  antitoxin 
itself,  but  are  to  be  attributed  exclusively  to  the  serum  of 
the  horse.  Like  amounts  of  antitoxic  serum  induce  the 
same  results  in  this  respect,  whether  they  contain  a  larger 
or  a  smaller  number  of  immunity-units.  For  this  reason  it 
is  desirable  to  obtain  as  highly  concentrated  an  antitoxin 
as  possible  in  order  that  as  small  amounts  of  serum  as  pos- 
sible need  be  injected.  Behring  succeeded  in  preparing  an 
antitoxin  of  1200  times  the  normal  strength,  but,  unfortu- 
nately, it  was  soon  found  that  such  highly  concentrated 
solutions  could  not  be  preserved  for  as  long  a  time  as  less 
concentrated  solutions,  and  that  in  the  course  of  time  the 
immunizing  activity  diminished  considerably. 

Behring  was,  therefore,  led  to  the  preservation  of  serums 
of  greater  than  500-strength  by  converting  them  into  a  dry 
form  by  a  special  procedure.     The  dry  powder,  containing 

*  These  results  have  been  amply  confirmed   not  only  on  the  Continent  of 
Europe,  but  also  by  observations  in  England  and  in  America,  with  reference  • 
to  both  nonoperative  and  operative  cases. — A.  A.  E. 


230  CLINICAL  BACTERIOLOGY. 

sodium  chlorid  and  albumin,  can  be  preserved  for  an  in- 
definite time  without  any  addition,  and  it  is  readily  soluble 
in  water.  One  gram  contains  at  least  5000  immunity-units, 
and  in  some  preparations  as  much  as  10,000  immunity- 
units.  From  Yt  ^^  y^  gram  thus  represents  a  simple  cura- 
tive dose  ;  from  ^  to  ^^  gram,  twice  as  much,  for  severe 
cases  ;  and  from  %  to  yi  gram,  four  times  as  much,  for 
advanced  cases.  Behring  considers  it  desirable  that  the 
preparation  of  these  solutions  should  be  undertaken  by 
pharmacists,  and  he  is  hopeful  that  the  dried  serum  may 
in  due  time  be  incorporated  into  the  Pharmacopeia. 

Prophylaxis. — It  is  important  to  know  that  after  com- 
plete disappearance  of  the  membrane  virulent  diphtheria- 
bacilli  may  persist  in  the  mouth  and  pharynx  of  children 
until  the  fifth  week,  and  sometimes  even  much  longer. 
The  children  must,  therefore,  be  isolated  for  from  five  to  six 
weeks  after  recovery  from  the  disease,  and,  above  all,  be 
kept  from  school  during  that  time.  Those  who  surround 
the  patient  should  also  be  kept  under  medical  observation. 
Careful  disinfection  of  the  sick-room  is  a  matter  of  impor- 
tance, as  it  is  known  that  diphtheria-bacilli  will  persist 
for  a  long  time  and  most  tenaciously  in  a  house  in  which 
there  has  been  a  case  of  diphtheria. 

With  regard  to  personal  prophylaxis  by  means  o^  injections 
of  antitoxin,  this  has  not  yet  been  employed  upon  a  large 
scale.  An  injection  of  250  normal  antitoxin-units  confers 
protection  lasting  three  or  four  weeks.  This  will  suffice  in 
most  cases,  but  if  the  danger  of  infection  continues  for  a 
longer  time,  a  second  prophylactic  injection  will  become 
necessary.  The  main  objection  that  has  hitherto  stood  in 
the  way  of  this  form  of  diphtheria-prophylaxis  was  the  fear 
of  disagreeable  complications  due  to  the  serum.  Such  fear 
would,  however,  be  removed  by  the  use  of  a  highly  con- 
centrated antitoxin  in  powder-form,  as  0.025  gram  of  the 
powder  with  a  strength  of  10,000  contain  the  requisite  250 
immunity-units. 


TETANUS. 

The  exciting  agent  of  tetanus  was  recognized  by 
Nicolaier,  of  Gottingen,  in  1885,  as  a  bristle-like  rod  with  a 
terminal  bulbous  spore,  but  it  was  first  isolated  by  Kitasato 
in  1889  from  the  foreign  bacteria  always  associated  with  it 


TETANUS.  231 

in  infectious  earth  and  in  pus  from  a  case  of  tetanus  and 
grown  in  pure  culture. 

The  tetanus-bacillus  is  a  long,  slender  rod,  from  0.3  to  0.5  /a 
thick  and  from  3  to  5  /a  long,  with  rounded  extremities  and  with 
feeble,  but  distinct,  motility,  which,  however,  ceases  immediately 
in  the  presence  of  oxygen.  It  frequently  grows  in  filaments, 
the  individual  elements  of  which  are  not  always  distinctly  dif- 
ferentiable  from  one  another.  The  temperature-optimum  is 
from  36°  C.  (96.8°  F.)  to  38°  C.  (100.4°  F.),  although  the 
bacillus  thrives  also  at  room-temperature.     It  does  not  develop 


FJg*  53.— Bacillus  tetani;   X  looo  (Friinkel  and  Pfeiffer). 

below  a  temperature  of  14°  C.  (57.2°  F.),  while  at  42°  C. 
Qio7.6°  F.)  or  43°  C.  (109.4°  F.)  it  presents  distinct  invo- 
lution-forms, and  at  60°  C.  (140°  F. )  it  is  destroyed  quite 
rapidly.  The  tetanus-bacillus  is .  an,  anaerobic  organism, 
although  it  still  grows  in  the  presence  of  small  amounts  of 
oxygen.  Protected  from  air  and  light  the  tetanus-spores  retain 
their  vitality  and  their  virulence  in  cultures  after  the  lapse  of  a 
year. 

Spore-formation. — At  a  temperature  of  37°  C.  (98.6°  F.) 
after  the  lapse  of  thirty  hours,  and  at  room-temperature  within  a 
week,  the  tetanus-bacillus  forms  spherical  spores  always  situated 
at  one  extremity.  The  bacillus  swells  at  one  extremity  like  a 
drumstick,  and  there  results  the  characteristic  head-bearing 
bacillus  (pin-shaped  or  note-shaped).     The  organism  is  always 


232  CLINICAL  BACTERIOLOGY. 

nonmotile.  The  diameter  of  the  spores  measures  from  i  to  i .  5  /a. 
They  are  extremely  resistant  to  the  action  of  heat.  They  are 
not  injured  by  exposure  for  an  hour  to  a  temperature  of  80°  C. 
(176°  F.),  and  they  are  not  destroyed  by  live  steam  with  a 
temperature  of  100°  C.  (212°  F. )  before  the  lapse  of  five  or 
eight  minutes.  The  spores  are  also  rather  resistant  to  chemic 
disinfectants.  They  are  not  destroyed  in  five  per  cent,  carbolic 
acid  before  the  lapse  of  fifteen  hours ;  and  they  die  in  one  per 
cent,  solution  of  mercuric  chlorid  within  three  hours. 

Staining. — The  tetanus-bacilli  stain  readily  with  the  usual 
dyes,  and  they  can  be  stained  also  by  Gram's  method.  The 
spores  can  be  demonstrated  by  the  usual  methods  for  staining 
spores. 

Pure  Cultures. — The  tetanus-bacillus  is  encountered  in 
nature  (garden-soil,  dust,  animal  excrement)  and  also  in  the 
pus  from  wounds  infected  with  tetanus  always  in  association 
with  numerous  other  bacteria,  some  of  which  are  anaerobic  and 
others  aerobic.  On  account  of  the  difficulty  of  isolating  the 
tetanus-bacillus  from  its  associates,  the  attempt  to  obtain  it  in 
pure  culture  long  failed.  Kitasato  overcame  this  difficulty  by 
availing  himself  of  the  great  resistance  of  the  tetanus-spores. 
He  inoculated  tetanus-pus  upon  agar-tubes ;  after  exposure  for 
two  days  in  the  thermostat,  in  addition  to  the  other  bacteria 
there  were  found  also  numerous  characteristic  bristle-like  rods 
with  bulbous  heads.  The  mixed  culture  was  now  heated  in  the 
water-bath  at  temperature  of  80°  C.  (176°  F. )  for  an  hour, 
when  all  of  the  bacteria,  including  the  tetanus-bacilli,  were  de- 
stroyed, and  only  the  tetanus-spores  survived.  These  could 
now  be  grown  in  pure  culture  without  difficulty  by  the  various 
culture-methods  employed  for  other  anaerobic  bacteria.  It 
ought,  however,  not  occasion  surprise  if  a  pure  culture  should 
not  succeed  even  by  this  method,  if,  as  happens  rarely,  other 
resistant  spores  are  present  in  the  material  submitted  to  exam- 
ination. Under  these  circumstances  anaerobic  plates  must  be 
made,  by  means  of  which,  further,  it  is  possible  to  isolate  the 
tetanus-bacilli  in  the  discharge  (pus). 

Cultural  Properties  of  the  Tetanus-bacillus. — The 
tetanus-bacillus  grows  upon  all  the  customary  culture-media 
with  exclusion  of  oxygen,  and  to  which  grape-sugar  (two  per 
cent.)  may  be  advantageously  added.  All  tetanus-cultures  pos- 
sess in  common  a  peculiar,  rather  disagreeable  odor  of  burned 
material. 

On  gelatin-plates  at  room-temperature  there  appear  about  on 
the  fifth  day,  slowly  growing,  small  colonies,  with  radiate  pro- 
cesses that  give  to  the  whole  a  feathery  or  thistle-like  appear- 
ance. Microscopic  examination  discloses  in  the  center  a  dense, 
yellowish  mass,  with  numerous  delicate,  ciliary  fibers  and  pro- 


TETANUS.  233 

cesses  arranged  radially  at  the  brighter  margin.  The  gelatin  is 
slowly  liquefied. 

In  high  gelatin  stab-cultures  growth  confined  to  the  lower  por- 
tion of  the  line  of  inoculation  appears  after  about  a  week. 
From  the  grayish-white  bacterial  mass  innumerable  small,  pointed 
processes  extend  in  every  direction  into  the  gelatin,  giving  the 
culture  a  characteristic  appearance  suggestive  of  a  large-branched 
fir-tree.  In  the  second  week  liquefaction  sets  in  and  obliterates 
this  appearance.  The  process  advances  slowly,  until  gradu- 
ally the  entire  culture  is  converted  into  a  turbid,  grayish-white, 
viscid  mass,  of  which  the  upper  portion  subsequently  becomes 
clear,  while  the  bacilli  sink  to  the  bottom  as  a  cloudy  gray  mass. 

On  agar-plates  delicate  colonies  develop,  which  with  low 
powers  of  the  microscope  appear  to  consist  of  a  network  of  fine 
threads  such  as  is  not  usually  seen  in  anaerobic  cultures. 

On  agar  stab-cultures  the  growth  is  similar  to  that  upon  gela- 
tin, although  not  so  pronounced.  It  takes  place  much  more 
rapidly  at  the  temperature  of  the  body.  Stab-cultures  in  a  high 
layer  of  grape-sugar  agar  develop  within  from  twenty-four  to 
forty-eight  hours  close  to  the  surface.  They  exhibit  the  character- 
istic odor,  and  are  usually  attended  with  abundant  gas-formation. 

In  glucose-bouillon  growth  at  37°  C.  (98.6°  F.)  is  very  ener- 
getic. On  account  of  the  abundant  formation  of  gas  it  is  well 
not  to  close  the  bouillon-flasks  too  tightly.  The  bouillon  is  at 
first  rendered  quite  turbid ;  after  standing  for  weeks  the  bac- 
terial masses  settle  to  the  bottom  as  a  grayish-white  layer,  so 
that  on  careful  suction  of  the  overlying  clear  fluid  a  solution  of 
toxin  free  from  bacteria  is  obtained.  This  may  be  secured  with 
greater  certainty  by  filtering  the  bouillon-cultures  through  porce- 
lain cylinders. 

The  tetanus-bacillus  develops  in  milk  without  causing  any 
change. 

Upon  potatoes  a  moist  invisible  deposit  forms  similar  to  that 
caused  by  typhoid-bacilli  (Vaillard  and  Vincent). 

Tetanus  in  Animals. — Tetanus  occurs  under  natural 
conditions  in  horses,  sheep,  and  neat  cattle  ;  it  has  not  been 
observed  in  dogs,  and  especially  not  in  fowl.  The  mouse  is 
the  animal  best  adapted  for  the  experimental  development 
of  tetanus.  Of  an  old  bouillon-culture  (freed  of  bacilli  by 
filtration,  or  by  careful  decanting)  o.ooi  cu.  cm.,  and 
frequently  still  much  smaller  amounts,  suffice  on  subcu- 
taneous injection  to  cause  death  in  white  mice  (from  twelve 
to  fifteen  grams  in  weight)  within  twenty-four  hours.  The 
guinea-pig  is  almost  equally  susceptible.  The  rabbit  is 
much  less  susceptible  :  not  less  than  from  o.  5  to  i  cu.  cm. 


234  CLINICAL   BACTERIOLOGY. 

of  the  culture  mentioned  being  required  to  cause  tetanus  in 
an  animal  weighing  about  looo  grams,  with  death  after  the 
lapse  of  several  days.  The  dog  may  be  looked  upon  as 
being  naturally  immune  to  tetanus,  being  affected  only  on  in- 
oculation with  large  doses  of  toxin  :  from  5  to  lo  cu.  cm.  and 
more.  Birds  are  highly  immune,  withstanding  inoculations 
of  from  10  to  20  cu.  cm.  of  highly  toxic  tetanus-bouillon  ; 
still  larger  amounts  of  toxin,  naturally,  give  rise  to  fatal 
tetanus  both  in  the  hen  and  in  the  pigeon.  Frogs  also 
may  suffer  from  tetanus  if  after  inoculation  they  are  kept 
permanently  in  a  heated  room  ;  but  relatively  large  doses 
of  toxin  are  necessary,  and  the  disease  develops  only  after 
two  or  three  weeks. 

Introduction  of  the  tetanus-virus  into  a  wound  or  injec- 
tion into  the  large  cavities  of  the  body  or  directly  into  the 
veins  acts  in  the  same  way  as  subcutaneous  inoculation. 
Experimental  infection  can  not  be  induced  by  way  of  the 
digestive  or  the  respiratory  tract. 

The  period  of  incubation  varies  in  accordance  with  the 
susceptibility  of  the  animal  and  the  virulence  and  amount 
of  toxin  inoculated  :  between  one  and  two  days,  in  the 
mouse  ;  and  between  eight  and  fourteen  days,  in  the  rabbit. 

Tetanus  in  animals  manifests  itself  in  the  form  of  extensor 
spasm,  which  gives  rise  to  a  clinical  picture  entirely  analo- 
gous to  that  exhibited  by  tetanus  in  human -beings.  The 
spasm  appears  first  in  the  neighborhood  of  the  site  of  inocu- 
lation ;  thus,  when  the  root  of  the  tail  is  inoculated,  in  the 
hind  extremities  and  also  in  the  tail ;  when  the  nape  of  the 
neck  is  inoculated,  in  the  fore  extremities  and  the  muscles 
of  the  neck.  In  the  further  development  of  the  disease  the 
entire  muscular  system  is  involved  in  the  spasm,  which 
quickly  leads  to  death.  After  intraperitoneal  and  intra- 
venous inoculation  the  spasm  is  general  from  the  outset. 
The  longer  the  period  of  incubation,  the  slower  generally 
is  the  course  of  the  disease,  and  the  shorter  the  period  of 
incubation,  the  more  unfavorable  is  the  prognosis.  After  the 
disease  has  existed  for  some  time  recovery  not  rarely  takes 
place.  This  ensues  always  but  slowly,  the  rigid  extremities 
relaxing  in  the  course  of  weeks  or  months.  On  postmortem 
examination,  only  slight  changes  are  found  at  the  site  of  in- 
oculation when  a  pure  culture  has  been  employed  in  the 
inoculation — viz.,  slight  infiltration  or  hemorrhage,  and 
nothing  else.     If  the  inoculation  has  induced  no  extensive 


TETANUS.  235 

injury,  the  site  of  inoculation  may  escape  observation  at 
autopsy.  If  a  mixed  culture  was  inoculated — e.  g.,  tetanus- 
infected  earth,  or  pus  or  fragments  of  tissue  from  a  wound 
in  a  case  of  tetanus — a  focus  of  suppuration  will  be  found 
at  the  site  of  inoculation.  No  changes  are  demonstrable  in 
the  remaining  organs  of  animals  dead  of  tetanus  ;  above  all, 
tetanus-bacilli  have  never  been  found  present,  and,  likewise, 
not  in  the  blood.  The  bacilli  have  been  demonstrated  only 
at  the  site  of  inoculation,  and  in  rare  instances  in  the 
nearest  adjacent  lymph-glands  ;  but  even  in  these  places 
they  are  present  only  in  small  number,  and  they  usually 
can  be  found  only  with  great  difficulty. 

The  Nature  of  Tetanus. — The  postmortem  findings  just 
described  are  explained  by  the  fact  that  tetanus  is  an  ex- 
quisitely toxic-infectious  disease.  The  convulsions  and 
death  are  due  to  a  poison  generated  by  the  tetanus-bacilli 
and  rapidly  absorbed  from  the  site  of  inoculation.  The 
evidence  in  favor  of  this  view  has  been  derived  from  vari- 
ous sources.  In  the  first  place,  exactly  the  same  clinical 
picture  can  be  developed  with  the  germ -free  filtrate  of  a 
tetanus  bouillon-culture,  which  thus  contains  only  the  dis- 
solved chemic  tetanus -toxin,  as  by  means  of  the  bacilli 
themselves.  In  the  next  place,  Kitasato  has  inoculated 
mice  with  tetanus-bacilli  at  the  root  of  the  tail,  and  has 
excised  and  cauterized  the  site  of  inoculation  throughout 
a  large  extent  after  one-half,  one,  and  one  and  a  half  hours, 
and  longer,  so  that  there  was  no  chance  for  the  bacilli, 
which  do  not  penetrate  beyond  the  site  of  inoculation,  to 
be  retained  within  the  body,  and  only  the  toxin  absorbed 
could  in  any  way  be  responsible  for  the  further  develop- 
ment of  the  disease.  Only  those  animals  operated  on  half 
an  hour  after  inoculation  remained  well,  while  all  of  the 
others  were  attacked  with  tetanus.  This  means  that  as 
early  as  an  hour  after  the  inoculation  so  much  toxin  was 
absorbed  that  the  bacilli  were  no  longer  necessary  to  the 
development  of  the  attack  of  tietanus. 

The  subordinate  position  taken  by  the  bacilli  in  their  sig- 
nificance for  the  clinical  picture,  as  compared  with  the 
toxins  to  which  they  give  rise,  led  Vaillard  and  Vincent  to 
the  conclusion  that  the  bacilli,  through  themselves  and  their 
spores,  are  not  alone  capable  of  causing  the  disease,  and  that 
in  a  pure  state  they  are  entirely  inactive  ;  and  only  the  layer 
of  toxin  which  adheres  to  them  externally  gives  rise  to  the 


236  CLINICAL  BACTERIOLOGY. 

outbreak  of  tetanus.  If  this  is  removed,  for  instance  by 
washing  with  large  amounts  of  water  or  by  exposure  to  a 
temperature  of  65°  C.  (149°  F.)  or  by  growth  of  the  cul- 
tures at  a  temperature  of  20°  C.  (68°  F.)  or  22°  C.  (71.6° 
F.) — under  which  conditions  the  bacilli  generate  no  poison 
for  the  first  six  days — such  toxin-free  bacilli  will  be  in- 
capable of  inducing  tetanus."  These  views  have  in  general 
been  confirmed.  The  introduction  of  toxin-free  bacilli  does 
not,  as  a  rule,  give  rise  to  infection  ;  but  the  spores  freed 
of  toxin  reacquire  their  virulence,  and  tetanus  will  develop 
with  certainty  if  with  them  there  are  introduced  other  bacte- 
ria not  giving  rise  to  tetanus,  or  if  simultaneously  trauma- 
tism is  inflicted,  or  a  splinter  of  wood  is  forced  beneath  the 
skin,  or,  finally,  a  chemic  agent — for  instance,  lactic  acid, 
trimethylamin,  etc. — is  injected  at  the  same  time.  These 
experimental  data  are  of  the  highest  importance  for  a  com- 
prehension of  tetanus  developing  spontaneously.  From 
them  the  probable  conclusion  may  be  drawn  that  under 
natural  conditions  tetanus  is  not  induced  by  the  tetanus- 
bacilli  alone,  but  that  the  presence  of  favoring  bacteria — 
thus  a  mixed  infection — is  necessary,  or  that  a  severe  trau- 
matism, or  the  like,  must  attend  the  infection. 

The  question  has  been  studied  experimentally  as  to  how 
the  extensor  spasm  arises  in  cases  of  tetanus,  and  whether 
the  tetanus-toxin  has  a  central  or  a  peripheral  action. 
Tizzoni  and  Vaillard  divided  all  the  nerves  of  an  extremity 
in  an  animal  before  inoculation  with  tetanus.  This  member 
remained  relaxed,  whereas  the  remainder  of  the  muscular 
system  became  rigid.  Buschke  curarized  a  tetanized  frog, 
and  the  tetanus  ceased  at  once.  The  toxin,  thus,  can  not 
act  upon  the  muscles  themselves  or  upon  the  peripheral 
nerves.  After  removal  of  the  brain  the  tetanized  frog 
remained  rigid.  A  direct  influence  of  the  poison  upon  the 
motor  centers  through  application  to  the  cerebral  cortex 
was  without  effect  upon  rabbits.  It  thus  appears  that  the 
brain  also  is  not  the  part  attacked  by  the  toxin.  Gradual 
destruction  of  the  spinal  cord  in  tetanized  animals  causes 
disappearance  of  the  rigidity  in  the  parts  corresponding  to 
the  respective  sections  of  the  cord.  Accordingly,  the 
activity  of  the  toxins  appears  to  be  localized  in  the  spinal 
cord,  as  is  the  case  also  with  strychnin. 

The  Tetanus- toxin. — The  germ-free  filtrate  of  a  teta- 
nus-culture  from    two    to    four  weeks  old  that  has   been 


TETANUS.  237 

grown  upon  feebly  alkaline  glucose-bouillon  contains  the 
specific  tetanus-toxin  in  an  exceedingly  active  state.  Often 
as  little  as  0.000005  ^u.  cm.  of  such  a  filtrate  will  suffice 
to  cause  death  in  a  white  mouse.  The  toxin  is  destroyed 
in  solution  by  exposure  to  a  temperature  of  65°  C.  (149° 
F.)  for  five  minutes,  and  it  is  gradually  enfeebled  in 
the  thermostat.  In  a  cold  compartment  (refrigerator)  and 
protected  from  light  it  retains  its  toxicity  unchanged  for 
months.  For  the  preservation  of  such  a  toxic  filtrate  it  is 
advisable  to  add  glycerin  in  equal  amount,  or  carbolic  acid 
in  proportion  of  0.5  per  cent.,  and  it  may  then  be  employed 
for  months  in  the  same  way  as  the  solution  of  a  chemic 
poison  of  known  strength. 

Efforts  have  not  been  wanting  to  isolate  the  toxin  in  a 
pure  state.  Brieger  obtained  from  cultures  two  basic  sub- 
stances— tetanin  and  tetanotoxin — both  of  which  caused 
death  in  animals,  preceded  by  manifestations  like  those  of 
tetanus.  Neither,  however,  can  have  any  important  bear- 
ing upon  the  occurrence  of  the  disease,  as  relatively  large 
amounts  were  required  in  order  to  induce  disease  in  animals. 
The  actual  tetanus-toxin,  however,  must,  in  accordance 
with  what  is  known  regarding  the  toxicity  of  the  bouillon- 
filtrate,  be  active  in  minimal  amounts.  Brieger  and  Frankel 
have,  according  to  their  method,  by  precipitation  with 
alcohol,  obtained  a  toxalbumin  that  is  much  more  active. 
Even  this  albuminous  powder  does  not  represent  the 
tetanus-toxin  chemically  pure ;  the  toxic  substance  is 
merely  adherent  to  it. 

The  attempts  to  obtain  the  virus  in  its  chemic  purity 
have  been  pursued  unremittingly,  and  it  has  been  obtained 
in  a  quite  concentrated  form.  Reference  was  made  in  the 
general  section  (p.  30)  to  the  investigations  of  Brieger  and 
Boer,  from  which  it  appears  that  the  toxin  of  the  tetanus- 
bacillus  is  not  an  albuminous  substance  at  all.  These 
investigators  precipitated  the  tetanus-toxin  by  the  original 
toxalbumin-method  of  Brieger  and  Frankel  with  the  aid 
of  ammonium  sulphate,  and  then  by  means  of  a  weak 
solution  of  mercuric  chlorid  (from  10  to  20  cu.  cm.  of 
a  0.05  per  cent,  solution  to  10  cu.  cm.  of  tetanus-toxin 
fluid)  precipitated  the  redissolved  toxin.  The  carefully 
washed  precipitate  is  well  rinsed  with  water,  and  decom- 
posed by  successive  treatment  with  ammonium  carbonate, 
ammonium  phosphate,  and  ammonium   sulphate.      Brieger 


238  CLINICAL  BACTERIOLOGY. 

and  Boer  conclude  the  description  of  this  rather  complicated 
procedure  with  the  following  statement :  If  the  readily- 
soluble  specific  toxin  has  been  cleansed  as  thoroughly  as 
possible  and  deposited  upon  the  filter,  it  will  be  completely 
absorbed  by  hardened  filters,  and  there  will  be  nothing  to 
suggest  its  presence  except  a  little  salt,  or  the  yellowish 
discoloration  of  the  filter,  which  is  often  quite  considerable, 
especially  in  the  case  of  the  tetanus-toxin.  The  activity  of 
these  substances  will,  therefore,  not  be  expressed  by  the 
sum  of  decimals,  but  to  a  certain  degree  only  as  integers. 
For  quantitative  studies  of  the  tetanus-toxin  the  bouillon- 
filtrate  appears  for  the  present,  therefore,  to  be  the  best 
suited. 

Tetanus-infection  in  Human  Beings. — The  portal  of 
entry  for  tetanus-bacilli  in  human  beings  may  be  con- 
stituted by  any  lesion  of  the  external  integument.  The 
tetanus-bacillus  has  been  repeatedly  demonstrated  in  the 
earth,  especially  in  that  which  has  been  manured,  and  more 
particularly  in  the '  uppermost  layers;  in  the  dust  of  the 
crevices  of  floors  ;  in  manure  from  horses  and  cows.  If  a 
certain  amount  of  any  of  these  substances  be  introduced 
subcutaneously  in  guinea-pigs,  there  result,  usually,  ma- 
lignant edema  and,  rarely,  tetanus.  This  result  depends 
upon  the  fact  that  the  bacilli  of  malignant  edema,  frequently 
present  in  earth  and  in  manure,  overrun  and  suppress  the 
tetanus-bacilli.  In  order  to  determine  whether  tetanus- 
bacilli  are  present  in  suspected  material  (earth,  etc.),  bouillon 
mixed  cultures  may  be  made,  according  to  the  suggestion 
of  Sanfelice,  and  kept  for  a  considerable  length  of  time  in 
the  thermostat,  and  then  filtered  through  porcelain  cylin- 
ders. The  results  of  injection  of  the  filtrate  will  decide 
whether  the  material  examined  contains  tetanus-bacilli 
or  not. 

Tetanus  in  human  beings  is  a  toxic  disease,  just  as  is 
tetanus  in  animals.  In  human  beings  also  the  bacilli  never 
enter  the  blood  or  the  viscera,  but  remain  confined  to  the 
site  of  original  infection.  On  the  other  hand,  the  presence 
of  the  tetanus-poison  in  the  blood  in  cases  of  tetanus  has 
been  demonstrated  by  successful  intoxication  of  mice  with 
the  blood-serum  from  such  cases.  Likewise,  a  substance 
has  been  obtained  from  the  liver,  the  spleen,  and  the  spinal 
cord  of  a  patient  dead  of  tetanus  by  precipitation  with  al- 
cohol, and  which,  dissolved  in  water,  is  capable  of  destroying 


TETANUS.  239 

small  animals  immediately,  after  the  development  of  tetanic 
manifestations.  Tetanus-toxin  has  been  found  on  several 
occasions  in  the  urine  of  tetanus-patients,  but  never  in  the 
sweat  or  in  the  saliva. 

The  portal  of  infection  is  ascertainable  in  the  majority 
of  cases.  Often  it  is  a  wound  of  considerable  extent,  which 
can  not  escape  observation — as  in  puerperal  tetanus  the 
wounded  surface  of  the  uterus,  in  tetanus  of  the  new-born 
usually  the  umbilical  wound,  in  cephalic  tetanus  (hydro- 
phobic tetanus)  an  injury  of  the  head.  The  source  of  infec- 
tion, also,  may  often  be  discovered  without  difficulty.  Not 
rarely  a  relation  between  the  patient  and  horses  can  be 
elicited,  as  these  animals,  according  to  Verneuil,  occupy  a 
central  position  in  the  etiology  of  tetanus  and  they  may 
convey  the  tetanus-bacillus  to  the  ground  with  their  manure  ; 
or  the  patient  may  have  introduced  beneath  the  skin  a 
splinter  to  which  the  bacilli  were  adherent ;  or  in  working 
about  a  garden  he  may  have  contaminated  an  already 
existing  wound  with  earth  ;  and  the  like. 

It  should  here  be  mentioned  that  tetanus-virus  retains 
its  virulence  for  an  exceedingly  long  time.  An  observation 
has  been  recorded  in  which  a  splinter  of  wood  that  had 
already  given  rise  to  infection  again  caused  tetanus  after 
the  lapse  of  eleven  years.  Bandages  and  dressings  from 
tetanus-wounds  likewise  preserve  their  infectivity  for  a  long 
period,  and  tetanus-cadavers  also  retain  for  a  considerable 
time  their  capability  of  causing  tetanus. 

Direct  contagion  has  also  been  observed,  tetanus  having 
been  transmitted  in  a  hospital-ward  from  one  patient  to 
his  neighbor,  or  several  patients  occupying  successively  the 
same  bed  each  developing  tetanus.  In  some  cases, 
however,  the  portal  and  the  mode  of  infection  are 
ascertainable  with  difficulty.  Cases  have  been  reported  in 
which  operation-wounds,  after  antiseptic  treatment,  healed 
by  primary  union  and  without  complication,  and  later' 
tetanus  developed  {cicatrix-tetanus).  Further,  there  occur 
cases  of  so-called  idiopathic  or  rheumatic  tetanus  in  which 
apparently  no  lesion  exists  anywhere,  and  also  at  autopsy 
no  suppuration  can  be  found.  The  information  gained  from 
experiments  on  animals  furnishes  a  complete  explanation  for 
these  cases.  The  portal  of  entrance  may  be  the  smallest 
abrasion  or  fissure  of  the  skin,  which  will  escape  observation, 
and  which  perhaps  has  already  healed  some  time  before  the 


240  CLINICAL  BACTERIOLOGY. 

convulsions  occur.  If  the  infection  is  single — that  is,  if 
pyogenic  cocci  do  not  gain  entrance  into  the  wound  at  the 
same  time  as  the  tetanus-bacilli — there  will  be  no  focus  of 
suppuration  at  autopsy  to  indicate  the  portal  of  infection. 
In  an  animal  experiment  recorded  by  Vaillard  spores  healed 
without  reaction  in  the  wound  induced  artificially,  and 
only  some  time  later,  when  the  affected  member  was 
irritated,  did  tetanus  occur.  The  cases  of  tetanus  that  occur 
in  connection  with  wounds  treated  antiseptically  are  suscep- 
tible of  a  similar  interpretation.  The  tetanus-bacillus  may 
long  remain  latent ;  the  antiseptic  employed  not  being  power- 
ful enough  to  destroy  the  tetanus-spores  with  which  the 
wound  is  infected,  and  these  are  included  in  the  healing 
wound,  to  proliferate  and  to  induce  the  disease  later  in  con- 
sequence of  some  predisposing  influence.  The  period  of 
incubation  of  tetanus  in  human  beings  varies  from  one  to 
twenty-two  days  ;  in  the  case  of  an  injury  induced  in  the 
laboratory  with  tetanus-toxin  it  was  four  days.  The  course 
of  the  disease  is  the  more  violent,  and  the  prognosis  the 
more  unfavorable,  the  shorter  the  period  of  time  that  has 
intervened  between  the  infliction  of  the  injury  (infection) 
and  the  outbreak  of  the  disease.  Recovery  took  place  in 
only  slightly  more  than  three  per  cent,  of  cases  with  an  in- 
cubation-period of  from  one  to  ten  days  ;  in  twenty-five  per 
cent,  of  those  with  an  incubation-period  of  from  ten  to 
twenty -two  days  ;  and  in  as  high  as  fifty  per  cent,  of  those 
with  longer  periods  of  incubation.  The  susceptibility  of 
human  beings  to  the  toxin  of  tetanus  must,  therefore,  be 
looked  upon  as  pronounced. 

Bacteriologic  Diagnosis. — If  it  is  desired  to  demonstrate 
the  bacilli  in  the  pus,  or  in  the  granulation-tissue  of  the 
wound  in  a  case  of  tetanus,  or  if  a  specimen  of  earth  or  a 
splintef  of  wood  is  to  be  examined  for  the  presence  of 
tetanus-bacilli  in  order  to  discover  the  source  of  infection, 
the  suspected  material  may  be  directly  introduced  into  a 
pocket  of  skin  at  the  root  of  the  tail  of  a  mouse.  If  the 
animal  dies  of  tetanus  in  the  course  of  a  few  days,  the 
pus  at  the  site  of  inoculation  is  treated  in  the  manner 
described  for  the  purpose  of  obtaining  a  pure  culture  (p. 
232).  The  material  to  be  examined  may  also  be  intro- 
duced into  bouillon,  through  which  a  current  of  hydrogen 
is  passed,  and  which  is  then  placed  in  the  thermostat  for 
from    three  to    five  days.      The    mixed    culture  that    has 


TETANUS.  241 

developed  in  the  bouillon  is  exposed  over  a  water-bath 
for  an  hour  at  a  temperature  of  80°  C.  (176°  F.),  and  from 
it  a  new  anaerobic  bouillon-culture  is  made  at  once.  If 
after  several  days'  development  this  culture  still  contains 
foreign  bacteria,  anaerobic  plates  are  prepared. 

Immunity  and  Cure  of  Tetanus  in  Animals. — The  laws 
of  immunity  and  immunization  have  been  more  thoroughly 
studied  in  tetanus  than  in  any  other  disease.  The  reason 
for  this  resides,  in  the  first  place,  in  the  exceedingly  sharp 
and  certain  reaction  of  animals  to  tetanus-infection,  and,  in 
the  second  place,  in  the  possibility  of  testing  quantitatively 
the  degree  of  immunity  by  means  of  treatment  with  the 
toxic  bouillon-filtrate. 

Immunity  to  a  toxic-infectious  disease  is  indicative  of 
proof  to  intoxication  :  an  animal  is  protected  from  infec- 
tion with  tetanus  if  the  tetanus-toxin  is  incapable  of  mani- 
festing its  toxic  activity  within  the  body  of  the  animal. 
Animals  slightly  susceptible  (dog,  hen)  can  be  further  im- 
munized simply  by  injection  of  gradually  increasing  amounts 
of  tetanus-toxin.  Their  serum,  in  the  hen,  likewise  the  egg- 
yolk,  also  acquires  thereby  immunizing  properties.  From 
the  serum  of  dogs  subjected  to  such  preliminary  treatment 
Tizzoni  and  Cattani  have  precipitated  their  so-called  anti- 
toxin by  means  of  alcohol. 

Behring's  method  of  immunization  by  means  of  tetanus 
bouillon-cultures  attenuated  with  iodin  trichlorid  is  applica- 
ble to  the  immunization  of  susceptible  animals  (mice,  rab- 
bits, horses,  sheep).  The  animals  are  treated  first  with 
a  bouillon-culture  containing  0.25  per  cent,  iodin  trichlorid, 
and,  finally,  with  undiluted  bouillon,  of  which  the  animals — 
smaller  at  intervals  of  from  three  to  five  days,  and  larger 
at  intervals  of  eight  days — then  receive  steadily  increasing 
doses,  until,  finally,  they  bear  quite  large  amounts  of  pure 
toxin. 

A  fluid  suitable  for  immunization  may  be  secured  also  by 
warming  the  cultures  (Vaillard  heats  the  filtrate  at  first  to 
60°  C— 140°  R,  later  to  55°  €.—131°  F.,  and,  finally,  to 
50°  C. — 122°  F.)  ;  further,  by  addition  of  iodin-water  or 
of  lactic  acid  to  the  cultures,  by  cultivation  of  the  bacilli 
in  thymus-bouillon,  etc. 

The  serum  of  immunized  animals  transmits  immunity  to 
animals  not  previously  treated ;  the    higher   the    original 
degree  of  immunity,  the  more  active  is  the  blood-serum. 
16 


242  CLINICAL  BACTERIOLOGY. 

This  acts  by  rendering  the  animal  toxin-proof;  it  contains 
an  antitoxin  that  enters  into  innocuous  combination  with 
the  tetanus-toxin. 

Also  after  infection,  and  even  after  the  development  of 
symptoms  of  tetanus,  the  serum  of  the  animal  may  afford 
protection  ;  it  thus  exerts  a  curative  action.  To  effect  cure 
in  animals,  however,  a  much  larger  amount  of  serum,  or  a 
much  more  highly  active  serum,  is  required  than  for  pro- 
phylactic immunization  ;  and  the  amount  of  serum  required 
becomes  the  greater  the  longer  the  period  of  time  that  has 
intervened  between  the  intoxication  and  the  employment 
of  the  serum.  According  to  observations  of  Donitz,  little 
more  serum  is  required  for  the  protection  of  the  animal 
four  minutes  after  the  intoxication  than  in  the  test-tube  for 
the  neutralization  of  the  amount  of  toxin  employed  (in  the 
test-tube  a  serum-dilution  of  i  :  2000,  in  the  body  after 
four  minutes  a  dilution  of  i  :  1 200) ;  after  the  lapse  of  eight 
minutes  six  times  the  amount  of  serum  will  be  required 
( I  :  200) ;  after  the  lapse  of  fifteen  minutes  twelve  times  the 
amount  (i  :  100) ;  and  after  an  hour  twenty -four  times  the 
amount  of  serum. 

Serum-therapy  in  Human  Beings. — It  is  obvious  that 
the  outlook  for  serum-therapy  in  the  case  of  tetanus  in  human 
beings  is  from  the  outset  less  promising  than  it  is  in  that 
of  diphtheria.  As  it  can  not  be  determined  from  inspec- 
tion that  a  wound  is  infected  with  tetanus-bacilli,  the  disease 
comes  under  medical  observation  only  after  the  toxin  has 
already  invaded  the  central  nervous  system,  and  has  induced 
more  or  less  extensive  and  in  part  perhaps  irreparable  injury. 
The  cases  of  tetanus,  therefore,  thus  far  treated  with  serum 
have  not,  on  the  whole,  yielded  good  results.  The  expla- 
nation for  this  may  be  found  in  the  weakness  of  the  serum ; 
in  view  of  the  lateness  of  application  of  the  treatment  the 
serum  should  be  of  especial  strength,  Behring  has,  there- 
fore, and  also  in  conjunction  with  Knorr,  sought  mainly  to 
increase  the  activity  of  the  serum.  To  obtain  tetanus-toxin 
he  employs  horses,  which  are  immunized  in  the  same  way 
as  to  diphtheria-toxin  (p.  218).  The  tetanus  normal  toxin, 
Tet  N  T^,  with  which  Behring  starts  out,  is  so  strong  that 
one  gram  contains  1,000,000,000  lethal  minimal  doses  for 
one  gram  by  weight  of  guinea-pig  (-f  1,000,000,000  M), 
1 50,000,000  lethal  doses  for  one  gram  by  weight  of  mice 
(-{-  150,000,000  Ms),  1,000,000  lethal  minimal  doses  for 


TETANUS.  ,  243 

one  gram  by  weight  of  rabbit  (-{-  1,000,000  K).  That 
serum  serves  provisionally  as  tetanus  normal  antitoxin, 
Tet  AN^,  whose  curative  activity  Behring  and  Knorr 
demonstrated  at  a  meeting  of  the  Physiologic  Society  in 
Berlin,  on  January  13,  1893.  This  serum,  injected  on 
several  days  successively  in  doses  of  0.04  cu.  cm.,  cured 
mice  that  had  been  infected  from  twenty -four  to  twenty- 
eight  hours  previously  with  the  lethal  minimum  dose,  and 
that  already  presented  distinct  symptoms  of  tetanus.  One 
cubic  centimeter  of  this  serum  contains  one  normal  anti- 
toxin-unit. For  the  future  it  is,  however,  probable  that 
that  serum  will  be  designated  as  normal  antitoxin  of  which 
I  cu.  cm.  will  render  innocuous  i  cu.  cm.  of  tetanus  nor- 
mal toxin. 

The  Hochst  Works  have  placed  two  preparations  on  the 
market.  The  first  occurs  in  the  form  of  a  dry  powder 
packed  in  5 -gram  vials.  This  antitoxin  has  100  times  the 
strength  "of  the  normal  (Tet  AN  ^^^)  ;  each  vial  thus  con- 
tains 500  tetanus  normal  antitoxin-units.  The  dried  serum 
keeps  well,  without  addition  of  an  antiseptic,  in  well-closed 
bottles,  and  remains  of  uniform  strength.  The  500  anti- 
toxin-units constitute  a  curative  dose  for  human  beings  and 
horses.  They  are  dissolved  in  45  cu.  cm.  of  sterile,  luke- 
warm water,  not  above  40°  C.  (104°  F.),  and  are  injected, 
in  the  case  of  horses,  directly  into  a  vein.  By  introduction 
into  the  blood-stream  a  much  more  energetic  action  is 
obtained,  and  this,  in  addition,  appears  twenty-four  hours 
earlier  than  after  subcutaneous  inoculation.  For  this 
reason,  Behring  and  Knorr  recommend  also  in  human 
beings,  in  urgent  cases,  that  the  injection  be  made  into  a 
vein.  Recovery  may  be  expected  after  subcutaneous  injec- 
tion in  acute  cases  only  when  the  serum  is  employed  before 
the  lapse  of  the  first  thirty -six  hours  after  the  symptoms 
of  tetanus  have  set  in.  The  time  lost  is  less  readily  com- 
pensated for  by  increase  of  the  dose  in  the  case  of  tetanus 
than  in  that  of  diphtheria. 

The  second  preparation  dispensed  by  the  Hochst  Works 
is  in  solution  in  vials  of  5  cu.  cm.  ;  i  cu.  cm.  contains  5 
normal  antitoxin-units — the  equivalent  of  five  times  the 
normal  antitoxin  (Tet  AN^).  As  in  the  case  of  the  diph- 
theria-antitoxin, o.  5  per  cent,  of  carbolic  acid  is  added  to 
prevent  decomposition.  This  dissolved  antitoxin  may  be 
employed  prophylactically  in  human  beings  and  animals 


244  CLINICAL  BACTERIOLOGY. 

after  such  injuries  as  experience  has  shown  may  be  followed 
by  tetanus.  The  size  of  the  dose  (from  0.5  to  5  cu.  cm.) 
is  governed  by  the  period  of  time  that  has  elapsed  since 
the  infliction  of  the  injury.  If  it  be  desired  to  make  an 
injection  of  antitoxin  in  advance  of  an  operation  that  is 
frequently  followed  by  tetanus  in  animals — as,  for  instance, 
preceding  castration — 0.2  cu.  cm.  will  suffice.  ' 

Recent  observations  have  afforded  noteworthy  support 
for  the  justification  and  the  utility  of  serum-therapy  with 
relation  to  tetanus.  Donitz  established  the  fact  in  the 
Institute  for  Serum-therapy  that  the  tetanus-toxin,  as  soon 
as  it  enters  the  blood,  is  taken  up  by  the  tissues  of  the  body, 
and  that,  in  cases  of  severe  intoxication,  the  simple  lethal 
dose  is  bound  within  from  four  to  eight  minutes.  The 
serum,  however,  separates  the  toxin  from  its  combination 
with  the  tissues,  and  neutralizes  it.  The  dissolution  of  the 
toxin-combination  is  effected  with  the  greater  difficulty  the 
severer  the  intoxication  and  the  longer  the  period  of  time 
that  has  elapsed  before  the  serum  is  employed.  Of  especial 
significance  is  the  discovery  by  Goldscheider  and  Flatau 
that  certain  degenerative  processes  affecting  nerve-cells  that 
are  caused  by  tetanus-intoxication  recede  under  the  influence 
of  the  serum.  We  have  thus  anatomic  evidence  for  the 
curative  activity  of  the  serum.* 

As  to  the  results  of  tetanus-therapy,  a  definite  opinion 
can  not  yet  be  given,  as  the  new  preparations  have  been 
generally  available  for  too  short  a  time.  As  bearing  upon  the 
value  of  prophylactic  inoculations  in  veterinary  medicine,  we 
have  the  statistics  of  Nocard,  covering  the  period  from 
August  I,  1895,  to  June  i,  1897.  Altogether,  2707  animals 
were  vaccinated.  Of  this  number,  2300  received  an  injec- 
tion of  serum  immediately  after  the  operation  (castration, 
amputation  of  the  tail,  etc.).  Not  one  of  these  animals 
developed  tetanus.  The  remainder  were  inoculated  from 
one  to  four  days  after  the  operation  or  after  the  infliction 
of  a  traumatism.  Only  one  horse  exhibited  symptoms  of 
tetanus,  but  these  rapidly  subsided.  Nocard  considers  these 
results  as  quite  remarkable,  as  the  animals  were  derived 
from  stables  in  which  tetanus  had  prevailed  shortly  before. 
On  the  other  hand,  the  sixty -three  veterinary  surgeons  who 

*The  results  appear,  further,  to  be  more  promising  when  the  serum  is  in- 
troduced beneath  the  dura  mater  than  after  any  other  method  of  introduction. 
—A.  A.  E. 


BOTULISM.  245 

had  furnished  Nocard  the  material  in  question,  observed 
259  cases  of  tetanus  in  the  same  time  in  animals  that  had 
not  been  inoculated. 


BOTULISM. 

In  spite  of  numerous  investigations  with  regard  to  the 
exciting  agent  of  meat-poisonirig  this  whole  question  was 
involved  in  considerable  doubt  until  recently,  when,  through 
the  discovery  by  Van  Ermengem,  in  an  epidemic  at  EUe- 
zelles  (Belgium),  of  a  specific  anaerobic  microbe,  the  bacillus 
botiillnus,  renewed  attention  was  directed  to  this  important 
subject. 

Under  the  designation  of  meat-poisoning  are  included 
two  entirely  distinct  symptom-complexes,  which  should 
properly  be  rigidly  differentiated  from  each  other.  The 
one  variety,  which  is  most  appropriately  designated  the 
gastro-intestinal,  simulates  the  clinical  picture  of  cholera 
nostras — a  simple  or  hemorrhagic  gastro-enteritis.  Among 
associated  manifestations  may  be  mentioned  fever,  albumin- 
uria, and  cutaneous  exanthemata  of  most  varied  kind  and  in- 
tensity. These  gastro-intestinal  lesions  arise  after  the  inges- 
tion of  decomposed  meat  or  of  meat  derived  from  diseased 
animals,  the  diseases  especially  concerned  being  pyemia, 
septicemia,  and  puerperal  fever.  In  most  cases  of  this 
kind  the  exciting  agents  are  bacteria  of  the  colon-group, 
and  in  other  cases,  occurring  less  commonly,  bacteria  of 
the  proteus-group. 

The  second  variety  is  identical  with  so-called  sausage- 
poisoning.  It  is  characterized  especially  by  nervous  symp- 
toms of  central  origin,  secretory  and  motor  disturbances, 
cessation  of  salivary  secretion,  dryness  and  redness  of  the 
mucous  membrane  of  the  mouth  and  pharynx,  dysphagia, 
hoarseness,  barking  cough,  paralysis  of  accommodation, 
mydriasis,  ptosis,  diplopia,  etc.  For  this  form  of  the  dis- 
ease the  designation  meat-poisoning  should  no  longer  be 
employed,  and  it  has  been  given  the  name  of  botulism. 

Botulism  may  arise  after  the  ingestion  of  special  kinds 
of  sausage — ^as,  for  instance,  blood-sausage  or  liver-sausage 
— which  are  prepared  particularly  in  certain  parts  of  Wiir- 
temberg  and  Baden.  It  is  further  caused  by  decomposed 
salt  fish,  by  smoked  meat,  ham,  preserved  meat,  venison, 
old  roasts,  and  the  like.     These  are  articles  of  food  that 


246 


CLINICAL  BACTERIOLOGY. 


are  intended  for  consumption  after  a  considerable  length  of 
time,  and  that,  according  to  Van  Ermengem,  are,  by  reason 
of  their  mode  of  preparation,  exposed  to  the  danger  of 
undergoing  anaerobic  fermentative  processes.  Van  Ermen- 
gem believes  the  actual  cause  to  be  the  anaerobic  bacillus 
botulinus,  which  he  isolated  in  the  epidemic  at  EUezelles 
from  a  piece  of  ham  that  gave  rise  to  typical  cases  of  in- 
toxication. As  opportunity  has  not  been  afforded  since 
Van  Ermengem  made  his  communication  for  investigating 
cases  of  botulism,  the  following  description  will  closely 
follow  his  statements. 


Fig.  54. — Bacillus  botulinus  ;  eight-day  culture  in  glucose-gelatin  ;  X  1000  (Van 
Ermengem). 


Morphology  of  the  Bacillus  Botulinus  (Fig.  54). — The 
bacillus  is  a  large,  slightly  motile  rod,  from  4  to  6, a  long  and  from 
0.9  to  1.2  ;tjt  thick,  with  slightly  rounded  extremities,  and  possess- 
ing from  4  to  8  flagella.  The  formation  of  filaments  is  but 
rarely  observed  ;  more  frequently  involution-forms  are  present, 
the  bacilli  becoming  smaller,  presenting  deficiencies,  and  being 
at  times  arranged  in  filaments.  Both  in  cultures  and  in  the 
body  the  bacillus  botulinus  generates  spores  of  oval  form,  gen- 
erally polar,  rarely  central,  and  exceeding  the  thickness  of  the 
bacillus.  The  bacillus  is  readily  stained,  and  also  by  Gram's 
method,  but  the  exposure  to  alcohol  must  not  be  too  protracted. 

Cultural  Peculiarities. — The  temperature-optimum  is  be- 
tween 20°  C.  (68°  F.)  and  30°  C.  (86°  F.).  Below  a  temper- 
ature of  16°  C.  (60.8°  F.)  the  bacillus  botulinus  grows  but 
slowly,  while  above  35°  C.  (95°  F.)  it  no  longer  generates 
spores,  does  not  itself  thrive  so  well,  and  exhibits  involution- 


BOTULISM.  247 

forms.  The  organism  is  strictly  anaerobic,  so  that,  in  order  to 
cultivate  it,  anaerobic  methods  of  investigation  must  be  pursued. 
The  culture-media  must  always  be  distinctly  alkaline,  and  the 
growth  is  favored  by  the  addition  of  two  per  cent,  of  grape- 
sugar. 

Gelatin-plates  exhibit  between  the  fourth  and  the  sixth  day 
round,  transparent,  brownish-yellow  colonies,  composed  of 
thick,  glistening  granules,  in  constant  movement.  The  colonies 
are  surrounded  by  a  slight  zone  of  liquefaction.  Later,  the 
margin  becomes  irregularly  radiate,  and,  finally,  processes  of 
the  most  varied  form  extend  out  from  it. 

High  gelatin  stab-cultures  exhibit  no  peculiarity.  Round, 
whitish  masses  develop  along  the  line  of  puncture,  at  times 
sending  processes  out  into  the  adjacent  gelatin.  The  gelatin  is 
not  liquefied,  but  active  gas-formation  takes  place. 

In  agar  stab-culture  the  growth,  apart  from  liquefaction,  is 
quite  similar. 

Glucose-bouillon  is  rendered  deeply  turbid. 

In  milk  slight  development  takes  place,  without  alteration  in 
the  nutrient  medium. 

On  potato  no  growth  takes  place. 

All  of  the  cultures  emit  an  odor  of  butyric  acid.  In  addition 
the  bacillus  generates  in  nutrient  media  containing  glucose  still 
other  fatty  acids,  butyl  alcohol,  hydrogen,  carbon  dioxid,  and 
methane. 


Tenacity  of  the  Bacillus  Botulinus. — The  cultures 
retain  their  capability  of  development  for  more  than  a  year 
if  they  are  kept  at  a  temperature  below  30°  C.  (86°  F.). 
At  temperatures  above  35°  C.  (95°  F.)  they  die  in  the 
course  of  a  few  weeks.  The  spores  possess  relatively  little 
resistance.  They  are  destroyed  at  a  temperature  approxi- 
mating 85°  C.  (185°  F.)  within  a  quarter  of  an  hour,  and 
at  a  temperature  of  80°  C.  (176°  F.)  with  certainty  within 
an  hour.  Five  per  cent,  carbolic  acid  causes  their  destruc- 
tion in  less  than  twenty-four  hours.  Dry  spores  exposed  to 
diffuse  daylight  exhibit  capability  of  germination  after  three 
months.  The  growing  forms  in  emulsion  with  distilled 
water  die  under  these  conditions  within  three  or  four 
weeks. 

Pathogenic  Properties. — The  bacillus  botulinus  proves 
pathogenic  when  fed  to  guinea-pigs,  mice,  and  monkeys. 
One  or  two  drops  of  a  liquefied  gelatin-culture  placed  upon  a 
bit  of  bread  or  in  milk  suffice  to  cause  the  death  of  the 
animal  within  one  or  two  days,  after  paretic  manifestations. 


248  CLINICAL  BACTERIOLOGY. 

mydriasis,  aphonia,  dysphagia,  and  blepharoptosis.  Cats 
withstand  the  action  of  considerable  amounts  of  the  cul- 
ture without  injury  when  introduced  by  the  stomach  ;  but 
subcutaneous  injection,  on  the  other  hand,  leads  to  death 
amid  such  typical  manifestations  that  the  cat  may  be  con- 
sidered as  the  physiologic  reagent  for  the  bacillus  botulinus. 
After  considerable  doses  (of  from  five  to  ten  cubic  centi- 
meters) cats  die  within  one  or  two  days,  but  after  smaller 
doses  (of  from  one  to  two  cubic  centimeters),  only  after 
from  eight  to  twelve  days.  After  an  incubation-period  gen- 
erally of  thirty-six  hours  the  animal  becomes  depressed,  no 
longer  moves  about,  and  refuses  nourishment.  On  the 
third  day  it  exhibits  a  peculiar  physiognomy.  The  facial 
expression  is  dull,  and  the  blinking  and  licking  move- 
ments that  healthy  cats  never  fail  to  exhibit  are  abol- 
ished ;  the  eyes  are  almost  completely  immobile,  and  the 
pupils  widely  dilated.  The  dilatation  of  the  pupils  be- 
comes, in  the  course  of  a  few  days,  quite  enormous.  The 
tongue  hangs  out  of  the  mouth  and  can  scarcely  be  drawn 
in  again.  Besides,  aphonia  sets  in  and,  further,  dysphagia, 
which  finally  may  increase  to  total  aphagia.  Urine  and 
feces  are  retained.  Death  occurs  usually  in  consequence 
of  paralysis  of  respiration  and  circulation.  Small  doses  of 
the  bacilli  give  rise  to  a  form  of  marasmus,  as  a  result  of  which 
the  cats  die  after  the  lapse  of  several  weeks  amid  paralytic 
manifestations  and  with  degeneration  of  the  parenchymatous 
organs.  Rabbits,  guinea-pigs,  and  mice  die  in  consequence 
of  subcutaneous  injection  of  minimal  amounts,  amid  pa- 
retic manifestations,  salivation,  dysphagia,  etc.  After  injec- 
tions of  one  or  two  cubic  centimeters  of  the  culture  pigeons 
exhibit  at  first  paresis  of  the  wings,  and  then  general  par- 
alysis. Intravenous  injection  leads  to  the  same  results  as 
subcutaneous  inoculation,  while  intraperitoneal  injection  is 
followed  by  less  marked  consequences. 

The  pathologic-anatomic  alterations  found  at  autopsy 
consist  in  more  or  less  marked  hyperemia  of  most  of  the 
viscera ;  in  an  acute,  sometimes  interstitial,  sometimes  par- 
enchymatous, hepatitis,  with  fatty  degeneration ;  in  des- 
quamative parenchymatous  nephritis ;  in  fatty  degenera- 
tion of  the  muscular  fibers  of  the  heart  and  of  the  ocular 
muscles.  Of  especial  interest  are  the  degenerative  changes 
in  the  central  nervous  system,  which  are  especially  marked 
in  the  spinal  cord,  and  less  so  in  the  medulla  oblongata. 


BOTULISM.  249 

These  are  entirely  wanting  in  the  central  nerves,  and  almost 
entirely  in  the  brain.  In  the  spinal  cord  the  gray  substance 
is  involved  almost  exclusively,  and  particularly  the  anterior 
horns ;  in  the  medulla  oblongata  the  nuclei  of  the  hypo- 
glossus,  the  dorsal  nucleus  of  the  vagus,  the  middle  small- 
cell  nucleus  of  the  oculomotor  nerve — in  short,  the  nuclei 
of  the  cerebral  nerves  affected. 

Physiology  of  Botulism. — Microscopic  examination  of 
the  organs  and  of  the  blood  of  animals  dead  of  botulism 
fails  to  disclose  the  presence  of  the  bacilli  anywhere  ;  even 
at  the  site  of  injection  they  are  to  be  found  in  only  small 
numbers  after  the  lapse  of  three  or  four  hours,  and  then 
distinctly  in  process  of  degeneration.  After  intravenous 
injection,  cultures  prepared  from  the  fresh  organs  exhibit, 
as  early  as  twelve  hours,  only  a  limited  number  of  colonies. 
If,  however,  these  organs  are  placed  in  the  thermostat  at  a 
temperature  of  30°  C.  (86°  F.),  numerous  microorganisms 
can  be  cultivated  from  them  after  the  lapse  of  from  twelve 
to  twenty-four  hours.  Botulism,  therefore,  represents  essen- 
tially an  intoxication  and  not  a  true  infection.  If  the  cul- 
tures are  freed  by  filtration  of  the  bodies  of  the  bacilli,  and 
if  animals  are  inoculated  with  the  toxic  solution  thus  ob- 
tained, precisely  the  same  morbid  manifestations  are  induced 
as  follow  inoculation  of  the  bacilli  themselves.  Van  Er- 
mengem  attempted,  in  the  same  way  as  Vaillard  and  Rouget 
in  the  case  of  tetanus,  to  free  the  spores  of  the  layer  of 
toxin  surrounding  them  by  washing  them  in  water,  and  he 
found  that  the  spores  thus  treated  were  much  less  active. 
He  was  unable,  however,  to  free  the  spores  entirely  of 
the  toxin,  and  he,  therefore,  came  to  the  conclusion  that 
the  protoplasm  of  the  microbes  must  retain  a  certain 
amount  of  toxin. 

Alkalies,  and  even  sodium  bicarbonate,  destroy  the  toxin 
of  botulism.  If  spores  are  permitted  to  macerate  for  a 
day  in  an  alkaline  solution  that  is  so  feeble  as  not  to 
destroy  their  germinating  capability — as,  for  instance,  a 
saturated  solution  of  sodium  bicarbonate — and  are  then 
exposed  for  two  hours  to  a  temperature  of  50°  C.  (122° 
F.),  and  animals  are  inoculated  therewith,  the  latter  continue 
to  live.  The  spores,  whether  introduced  under  the  skin  or 
into  the  stomach,  are  incapable  of  multiplying  and  generating 
toxin  ;  outside  the  living  body,  however,  the  spores  retain 
the  capability  of  generating  their  toxin  in  ordinary  strength.. 


250  CLINICAL  BACTERIOLOGY. 

Cultures  made  with  them  also  prove  equally  toxic  with 
those  from  spores  not  exposed  to  the  action  of  an  alkaline 
solution  and  of  heat.  The  influence  to  which  is  due  the 
fact  that  the  bacillus  botulinus  is  incapable  both  of  multi- 
plying and  of  generating  toxic  metabolic  products  within 
the  animal  body  is  not  yet  thoroughly  understood.  Per- 
haps it  is  due  to  a  high  degree  of  saprophytism,  which 
does  not  permit  the  microbe  to  adapt  itself  to  the  animal 
body  ;  and,  perhaps,  further,  to  the  fact  that  the  bodily  tem- 
perature, which  is  above  35°  C.  (95°  F.),  is  not  favorable 
to  its  development.  It  has  been  shown  experimentally  that, 
at  a  temperature  of  37°  C.  (98.6°  F.)  and  above,  involution- 
forms  occur,  and  no  toxin  is  generated.  The  bacillus  botu- 
linus thus  occupies  a  position  of  its  own.  It  is  pathogenic 
for  human  beings  only  by  reason  of  its  toxin,  which  it  forms 
outside  the  body  upon  dead  substances  (articles  of  food). 
Van  Ermengem  proposes  that  it  be  designated  as  toxico- 
genic. 

The  toxin  of  botulism  is  to  be  placed  upon  the  same 
plane  as  that  of  diphtheria  and  that  of  tetanus.  It  can  be 
obtained  by  precipitation  with  alcohol,  tannic  acid,  and 
neutral  salts.  It  was  further  separated  by  Brieger  and 
Kempner  by  treating  the  toxin-containing  filtrate  of  the 
culture  according  to  the  method  proposed  by  Brieger  and 
Boer  for  diphtheria  and  tetanus. 

Occurrence  of  the  Bacillus  Botulinus. — The  organism 
has  hitherto  never  been  observed  apart  from  its  discovery 
by  Van  Ermengem  in  ham  and  in  the  body  of  a  patient 
dead  of  botulism. 

Mixed  Infection. — The  associated  bacteria  that  were 
found  together  with  the  bacillus  botulinus  appear  neither 
to  favor  nor  to  inhibit  the  generation  of  toxin. 

Bacteriologic  Diagnosis. — Articles  of  food  that  have 
given  rise  to  botulism  are  examined  by  means  of  culture 
(plate -procedure)  for  the  presence  of  anaerobic  bacteria. 
In  the  case  of  Van  Ermengem  the  spores  of  the  bacillus 
botulinus  could  be  demonstrated  in  the  ham  microscopically. 
These  were  present  principally  in  the  red  portion  of  the 
ham,  and  in  smaller  number  in  the  fat.  They  were 
irregularly  distributed,  and  were  entirely  absent  in  some 
places.  In  addition,  it  is  well  to  inoculate  animals  with  an 
aqueous  maceration  (four  parts  of  chopped  ham  and  five 
parts  of  water).      In  the  bacteriologic  examination  of  the 


TUBERCULOSIS.  251 

bodies  of  individuals  dead  of  botulism  or  of  animals  that 
have  succumbed  to  injection  of  the  maceration,  it  is  to 
be  borne  in  mind  that  the  viscera  and  the  blood  contain  the 
bacilli  only  in  small  numbers.  It  is,  therefore,  necessary 
to  examine  large  quantities,  or  to  place  entire  organs  in  the 
thermostat  at  a  temperature  of  from  20°  C.  (68°  F.)  to 
30°  C.  (86°  F.),  in  order  to  bring  about  fertilization.  Van 
Ermengem  has  succeeded  in  isolating  the  bacillus  from  the 
spleen  and  from  the  gastric  and  intestinal  contents  from  the 
body  of  a  patient  dead  of  botulism. 

Immunity  and  Specific  Therapy. — Immunization  of 
animals  to  botulism  was  effected  by  Kempner  with  the 
filtrate  of  a  bouillon-culture  in  the  same  way  as  in  the  case 
of  diphtheria  and  of  tetanus.  The  serum  of  immunized 
animals  is  highly  antitoxic — a  further  evidence  of  the  fact 
that  the  toxin  of  botulism  is  closely  related  to  that  of 
diphtheria  and  that  of  tetanus.  When  Kempner  injected 
his  serum  in  doses  of  from  one  to  five  cubic  centimeters 
within  from  three  to  twenty-four  hours  after  inoculation  of 
guinea-pigs,  the  animals  survived.  As  investigations  of 
Kempner  and  Pollack  show,  changes  in  the  central  nervous 
system  have  already  taken  place  after  the  intoxication  has 
existed  for  twenty-four  hours.  These  changes  in  the 
nerve-cells,  as  the  anatomic  evidence  seems  to  show,  are 
neutralized  by  the  serum. 


TUBERCULOSIS. 

The  exciting  agent  of  all  tuberculous  processes  is  the 
tubercle-bacillus  discovered  and  cultivated  by  Robert  Koch 
in  the  year  1882. 

The  first  evidence  of  the  infectious  nature  of  tuberculosis  was 
furnished  in  1865,  by  Villemin,  who  rendered  healthy  animals 
tuberculous  by  inoculation  of  tuberculous  material.  These  ob- 
servations were  confirmed  by  Cohnheim,  and  they  were  supple- 
mented by  inoculation  into  the  anterior  chamber  of  the  eye. 
Cohnheim,  upon  the  basis  of  his  experiments,  formulated  the 
doctrine  of  the  specific  etiology  of  tuberculosis,  and  this  opinion 
was  crystallized  into  absolute  certainty  by  the  classic  bacteri- 
ologic  investigations  of  Koch  (**Arbeiten  aus  dem  Reichsge- 
sundheitsamt,"  11). 

Morphology  and  Staining  of  Tubercle-bacilli. — The 
tubercle-bacilli   are  delicate,   slender  rods,  from  0.2  to  0.4 /a 


252  CLINICAL   BACTERIOLOGY. 

thick,  and,  on  the  average,  from  3  to  4  ix  long.  They  are  slightly 
curved,  nonmotile,  and  as  a  rule  lie  singly,  but  in  cultures  at 
times  in  small  chains  of  from  4  to  6.  In  rare  cases  they  exhibit 
bulbous  terminal  enlargement  and  bifurcation,  in  consequence 
of  which  a  certain  relationship  with  the  actinomyces-group 
(p.  25)  is  suggested.  Tubercle-bacilli  are  distinguished  from  all 
other  bacteria  by  the  fact  that  they  stain  with  extreme  difficulty, 
but,  having  once  taken  up  the  stain,  they  retain  it  with 
great  tenacity.  The  simple  solutions  of  aniline  dyes  ordinarily 
employed  for  staining  other  bacteria  do  not  suffice  for  tubercle- 
bacilli  unless  the  exposure  be  very  prolonged.  For  this  reason 
tubercle-bacilli  are  stained  with  aniline-water  staining  solutions 
(Koch-Ehrlich  stain)  or  with  the  more  commonly  employed 
carbolfuchsin  (Ziehl  stain). 

Upon  cover-slip  preparations,  made  in   the  usual  way  from 
pure  cultures  and  fixed,  freshly  prepared  aniline-water  fuchsin 


/ 


^ 


1 


\ 


Fig-  55- — Tubercle-bacilli:  i,  Forms  suggesting  sporulation ;  2,  forms  described 
as  beaded  (the  open  spaces  in  the  fragmented  rods  are  sometimes  mistaken  for 
spores). 

(or  aniline-water  gentian-violet  or  aniline-water  methyl-violet), 
or  carbolfuchsin  is  dropped  ;  then  heat  is  applied  by  means  of  a 
small  gas-flame  or  spirit-flame  until  vapor  of  steam  arises,  and 
after  a  minute  the  excess  of  stain  is  washed  off  with  water.  The 
tubercle-bacilli  are  now  stained,  and  if  the  specimen  is  treated 
for  several  seconds  with  dilute  acid — for  instance,  fifteen  or 
twenty  per  cent,  nitric  acid — and  alcohol — from  60  to  70  per 
cent. — they  will  not  yield  up  their  color.  Stained  in  this  way 
the  tubercle-bacilli  frequently  exhibit  bright  deficiencies  that 
have  not  taken  the  stain.  These  were  at  first  looked  upon  as 
spores,  and,  later,  as  degenerative  manifestations,  but  both  views 
are  incorrect. 

Originally,  the  tubercle-bacilli  were  stained  in  cold  solutions, 
in  which  they  were  permitted  to  remain  for  several  hours ;  but 
by  application  of  heat  the  duration  of  the  exposure  can  be  re- 
duced to  a  few  minutes. 

The  cause  of  this  characteristic  behavior  in  staining  was  con- 


TUBERCULOSIS.  253 

sidered  by  Ehrlich  to  depend  upon  the  fact  that  the  tubercle- 
bacilli  possess  an  especially  resistant  cell-memdrane,  which 
permits  the  stain  to  enter  the  body  of  the  cell  only  with  the 
aid  of  mordants  (aniline  water,  carbolic  acid,  etc.).  This 
membrane,  later  in  the  process  of  decolorization,  prevents  the 
entrance  of  acids  into  the  interior  of  the  bacillus,  so  that  the 
microorganisms  do  not  give  up  their  stain.  The  tubercle-bacil- 
lus can  be  stained  by  Gram's  method. 

According  to  recent  investigations  by  Robert  Koch,  the 
tubercle-bacilli  contain  two  unsaturated  fatty  acids,  one  of  which 
is  soluble  in  dilute  alcohol,  and  is  saponified  by  sodium  hy- 
droxid  ;  whereas  the  other  is  not  saponifiable,  and  is  soluble  only 
in  boiling  absolute  alcohol  and  ether.  Both  fatty  acids  take 
the  specific  stain  of  the  tubercle-bacilli ;  but  as  one  of  them  is 
soluble  in  alcohol,  only  the  other  remains  after  decolorization, 
and  this  fixes  the  stain  and  must  be  looked  upon  as  the  medium 
of  the  color-reaction.  By  means  of  hot  sodium  hydroxid  the 
fatty  acids  may  be  slowly  driven  out  of  the  bodies  of  the  bacilli 
and  their  escape  in  the  form  of  tingible  drops  uniting  into 
larger  drops  can  be  observed  under  the  microscope.  According 
to  Koch,  these  fatty  acids  form  a  coherent  layer  within  the 
bodies  of  the  bacilli,  to  which  they  afford  protection  against 
external  influences. 

Culture  of  Tubercle-bacilli. — Pure  culture  of  tubercle- 
bacilli  is  difficult,  and  mainly  because  the  microorganisms 
develop  very  slowly  and  require  a  temperature  of  37°  C. 
(98.6°  F.)  for  their  growth.  Their  temperature-minimum 
is  29°  C.  (84.2°  F.),  their  temperature-maximum  41°  C. 
( 105.8°  F.),  and  their  temperature-optimum  37°  0.(98.6°  F.)  or 
38°  C.  (100.4°  F. ).  Tubercle-bacilli  thrive  well  upon  blood- 
serum,  upon  from  four  to  six  per  cent,  glycerin -agar,  and  upon 
glycerin-bouillon.  The  preparation  of  glycerin-agar  plates  for 
the  purpose  of  isolating  tubercle-bacilli  from  the  mixture  of 
bacteria  in  tuberculous  sputum  is  scarcely  possible,  as  the  tubercle- 
bacilli  develop  so  slowly  that  they  are  overgrown  and  suppressed 
by  the  colonies  of  the  other  bacteria.  Their  cultivation  will, 
therefore,  be  successful  only  if  the  material  examined  is  uncon- 
taminated.  To  this  end  the  following  plan  of  procedure  is 
pursued  :  Several  guinea-pigs-^animals  that  are  extremely  sus- 
ceptible to  tuberculosis — are  inoculated  with  material  containing 
tubercle-bacilli.  After  the  lapse  of  about  four  weeks  the  first  of 
the  inoculated  animals  will  die,  the  autopsy  revealing  marked 
tuberculosis  of  the  abdominal  viscera.  One  of  the  remaining 
guinea-pigs  is  now  killed,  its  skin  cleansed  most  carefully  by 
means  of  hot  water  and  i  :  1000  solution  of  mercuric  chlorid, 
the  skin  turned  back  with  a  knife  sterilized  in  the  flame,  the 
peritoneum  opened  with  another  instrument  similarly  treated. 


254  CLINICAL  BACTERIOLOGY. 

and  the  spleen  is  drawn  forward  with  forceps  sterilized  in  the 
flame,  as  this  organ  appears  to  be  involved  in  greatest  degree  in 
this  mode  of  infection.  A  bit  of  the  spleen  containing  a  tuber- 
culous nodule  is  excised  with  sterilized  scissors,  the  nodule  is 
compressed  between  two  aseptic  scalpels  or  glass  slides,  in  order 
to  set  the  tubercle-bacilli  free,  and  the  material  thus  obtained  is 
transferred  by  means  of  a  strong  platinum  wire  to  the  surface  of 
blood-serum  tubes.  The  whole  procedure  must  be  carried  out 
with  the  utmost  celerity  and  with  the  most  scrupulous  cleanliness, 
for  should  a  foreign  microorganism  gain  entrance  into  the  serum- 
tube,  it  will  soon  overgrow  the  tubercle-bacilli.  For  the  vSake  of 
greater  security,  several  tubes  are  always  treated  in  the  manner 
described.  As  the  tubes  must  be  kept  for  a  long  time  in  the 
thermostat  at  a  temperature  of  37.5°  C.  (99.5°  F.),  they  are 
closed  with  rubber  caps  that  have  been  sterilized  in  mercuric- 
chlorid  solution.  After  the  lapse  of  fourteen  days,  if  the  cul- 
ture be  successful,  the  first  signs  of  growth  are  observable  in  the 
serum-tubes.  In  the  neighborhood  of  the  expressed  material 
there  form  gray,  dry,  small  scales,  which,  with  low  powers  of 
the  microscope,  appear  to  be  made  up  of  delicate  curved  lines. 
Development  then  progresses  slowly,  and  after  the  lapse  of  from 
four  to  six  weeks  it  is  possible  to  continue  inoculations  from  this 
culture.  For  further  culture  blood-serum  tubes  likewise  are 
used.  Also  in  this  second  generation  growth  is  first  observed 
distinctly  only  after  the  lapse  of  two  weeks. 

Subsequent  generations,  usually  after  the  fifth  or  sixth,  grow 
more  vigorously  and  more  rapidly;  and,  particularly  when  a 
special  thermostat  is  employed  and  is  saturated  with  steam,  so  that 
the  rubber  caps  can  be  dispensed  with,  the  entire  surface  of  the 
serum,  after  from  seven  to  fourteen  days,  is  found  strewn  with  the 
characteristic  dry  scales.  From  the  fifth  serum-generation  trans- 
ference to  glycerin  nutrient  media  may  be  readily  effected. 

Upon  gfycerin- agar  develoipment  is  much  more  abundant  than 
upon  blood-serum.  The  bacilli  form  upon  this  a  grayish,  dry 
deposit  of  coarse  particles,  with  the  same  wavy,  slightly  raised 
outline.  This  coating  extends  downward,  and  envelops  the 
water  of  condensation  present,  without,  however,  rendering  it 
turbid  ;  and  if  the  culture  is  kept  long  enough  in  the  thermostat, 
the  deposit  grows  even  upon  the  free  surface  of  the  test-tube, 
where  no  nutrient  medium  is  present,  and  for  a  considerable 
distance  upward. 

As  a  fluid  culture-medium  veal-bouillon  with  six  per  cent,  gly- 
cerin is  preferably  selected,  and  this  is  poured  into  Erlenmeyer 
flasks.  In  inoculating  bouillon  the  dry  scales  must  be  intro- 
duced into  the  nutrient  fluid  in  such  a  way  that  they  float  upon 
its  surface.  The  tubercle-bacilli  have  a  strong  avidity  for 
oxygen,  and  they  develop  luxuriantly  only  where  air  has  suffi- 


TUBERCULOSIS.  255 

cient  access.  Upon  veal  glycerin-bouillon  the  tubercle-bacilli 
grow  in  the  form  of  a  superficial  membrane,  which  exhibits  the 
same  characters  as  the  deposit  upon  glycerin-agar.  In  this  also, 
after  the  lapse  of  weeks — under  favorable  conditions,  after  ten 
days — they  reach  the  walls  of  the  vessel,  and  likewise  grow  for 
a  certain  distance  upward  upon  the  glass.  The  underlying 
bouillon  remains  perfectly  clear — a  feature  that  is  characteristic 
of  the  growth  of  tubercle-bacilli. 

Upon  potatoes  whose  lower  extremity  projects  into  a  solution 
of  glycerin  (5  per  cent.)  and  sodium  chlorid  (0.5  per  cent.),  the 
tubercle-bacilli  grow  exceedingly  well.  Upon  the  surface  of 
the  potato  they  form  a  dense  deposit.  The  glycerin-solution 
remains  clear,  and  it  is  covered  by  the  well-known  coating.  In 
the  preparation  of  such  cultures  it  is  best  to  employ  the  cyl- 
inders of  potato  recommended  by  Roux. 

In  the  year  1892  Kitasato  described  a  procedure  suggested  by 
Koch  for  obtaining  tubercle-bacilli  directly  from  the  sputum  of 
tuberculous  patients.  The  patient  should  rinse  his  mouth  care- 
fully with  an  antiseptic  gargle,  and  then  expectorate  into  a  ster- 
ilized double  dish.  One  of  the  masses  of  sputum  ejected  is  washed 
in  several  sterilized  dishes  containing  sterile  water,  and  is  thus 
freed  from  the  bacteria  adherent  to  its  surface.  From  the  central 
portion  of  such  a  mass  a  flake  is  removed,  by  means  of  a  platinum 
wire,  and  smeared  upon  blood-serum  in  tubes.  The  cultures  that 
develop  under  these  conditions  exhibit  quite  a  different  appear- 
ance than  do  those  cultivated  from  the  animal  body.  There 
develop  round,  and  rather  whitish  and  translucent  colonies, 
which  in  subsequent  generations  grow  in  precisely  the  same 
manner  as  the  bacilli  obtained  by  the  method  first  described. 

Resistance  of  the  Tubercle-bacilli. — Tubercle-bacilli 
are  destroyed  only  after  exposure  for  ten  minutes  to  a  tem- 
perature of  70°  C.  (158°  F.)  ;  for  one  minute,  at  a  temper- 
ature of  95°  C.  (203°  F.)  ;  for  an  hour,  at  a  temperature 
of  60°  C.  (140°  F.) ;  and  for  four  hours,  at  a  temperature 
of  55°  C.  (131°  F.).  They  are,  thus,  more  resistant  to 
heat  than  most  other  varieties  of  vegetative  bacteria,  which 
are  generally  destroyed  by  brief  exposure  to  temperatures 
between  54°  C.  (129.2°  F.)  and  60°  C.  (140°  F.).  Tubercle- 
bacilli  resist  for  only  a  relatively  short  time  the  influence 
of  direct  sunlight — in  accordance  with  the  density  of  the 
bacterial  mass,  from  several  minutes  to  several  hours. 
Diffuse  daylight  causes  death  after  the  lapse  of  a  week. 
Tubercle-bacilli  can  be  cultivated  for  many  years  through 
a  series  of  generations,  without  loss  of  vitality,  although 
they  gradually  become  somewhat  less  virulent. 


256  CLINICAL  BACTERIOLOGY. 

Spontaneous  and  Experimental  Tuberculosis  in  Ani- 
mals.— Spontaneous  tuberculosis  occurs  in  animals,  with 
frequency  only  among  cattle  and  in  monkeys  living  in 
captivity,  although  there  is  scarcely  a  domestic  animal  that 
is  immune  to  tuberculosis.  Among  laboratory-animals  the 
guinea-pig  is  the  most  susceptible,  a  few  tubercle-bacilli 
sufficing  to  induce  infection  in  them.  Next  in  suscepti- 
bility is  the  rabbit,  and  then  the  field-mouse.  Less  sus- 
ceptible, though  by  no  means  immune,  are  white  mice  and 
dogs.  Young  animals  exhibit  a  much  greater  predisposi- 
tion to  tuberculosis  than  old  animals.  Typical  tubercu- 
lous disease  is  induced  experimentally  in  animals  (guinea- 
pigs,  rabbits,  field-mice)  by  means  of  subcutaneous  injec- 
tion, by  inoculation  of  the  anterior  chamber  of  the  eye,  by 
intrapleural,  intraperitoneal,  and  intravenous  injection,  or 
by  inhalation  of  moist  tubercle-bacilli  in  the  form  of  dust. 
The  last  remaining  portal  of  entry  for  tubercle-bacilli  into 
the  organism,  the  gastro-intestinal  tract,  also  was  utilized  suc- 
cessfully by  Koch  in  experiments  on  animals.  Susceptible 
animals  that  are  given  tubercle-bacilli  in  considerable  num- 
ber with  their  food  die  of  intestinal  tuberculosis.  The  re- 
sulting tuberculous  process  is,  apart  from  the  results  of 
intravenous  injection,  at  first  always  local,  restricted  to 
the  site  of  infection ;  it  extends,  however,  steadily,  but 
slowly,  by  way  of  the  lymph-paths.  The  bacilli  quickly 
gain  entrance  into  the  lymph-glands  nearest  the  portal  of 
infection.  As  early  as  three  days  after  inoculation  of 
tuberculous  material  into  the  anterior  chamber  of  the  eye 
Baumgarten  found  that  the  bacilli  had  advanced  as  far  as 
the  auricular  lymph-glands.  When  the  bacilli  are  injected 
into  a  vein,  general  miliary  tuberculosis  takes  place  at  once. 

Of  great  assistance  in  the  comprehension  of  some  tuber- 
culous local  processes  in  human  beings  are  the  observations 
of  Schiiller,  who  inoculated  tuberculous  material  at  some 
indifferent  portion  of  the  body  of  the  animal  and  then  in- 
flicted traumatism  in  the  neighborhood  of  the  knee-joint, 
and  found  that  the  infection  localized  itself  at  this  point. 

The  chronic  cold  abscesses,  which  upon  bacteriologic  ex- 
amination are  found  free  from  pyogenic  microbes,  are  cer- 
tainly to  be  attributed  to  tubercle-bacilli.  Dead  tubercle- 
bacilli,  destroyed  by  live  steam  or  by  other  means,  exhibit 
pyogenic  activity  experimentally.  They  are  positively 
chemotactic  :  they  attract  leukocytes.     The  pyogenic  sub- 


TUBERCULOSIS.  257 

Stance  contained  within  the  bodies  of  the  bacilli  can  be 
extracted  only  with  great  difficulty. 

If  dead  tubercle-bacilli  are  inoculated  into  rabbits  by  in- 
travenous injection,  and  the  animals  are  subsequently  killed 
(some  of  them  die  spontaneously),  the  lungs  and  the  liver  are 
found  strewn  with  small  nodules,  consisting  of  round  cells 
and  epithelioid  cells,  giant-cells,  which  contain  dead  tubercle- 
bacilli,  and  which  are  thus  indistinguishable  from  true 
tubercles.  Prudden  and  Hodenpyl,  who  were  the  first  to 
make  these  observations,  believed  the  formation  of  the 
tubercle  to  be  a  result  of  the  activity  of  a  specific  bacterial 
proteid  contained  within  the  protoplasmic  body  of  the  tuber- 
cle-bacilli. Baumgarten  does  not  agree  with  this  opinion, 
considering  the  formation  of  nodules  by  the  dead  bacteria 
as  tuberculosis  due  to  a  foreign  body  (p.  276).  The  agency 
through  which  tubercle-bacilli  in  general  give  rise  to  the 
formation  of  tubercles,  and  whether  a  toxic  influence  is 
operative,  are  questions  that  also  have  not  yet  been  deter- 
mined with  certainty  with  regard  to  the  living  bacilli.  It 
is  undoubted,  however,  that  the  tubercle  is  a  direct  result 
of  the  presence  of  the  tubercle-bacillus,  for  this  -is  found 
in  every  tubercle,  and  wherever  it  is  introduced  into  animals, 
there  tubercles  develop  with  certainty.  It  is  noteworthy 
that  in  the  clinical  picture  of  tuberculosis  manifestations 
of  general  intoxication  are  not  conspicuous  so  long  as  the 
process  is  in  its  incipiency,  is  localized,  and  mixed  infec- 
tion has  not  yet  taken  place. 

Pathologic  Anatomy  of  Tuberculosis  in  Animals. — 
In  different  varieties  of  animals  the  bacilli  cause  anatomic 
changes  of  a  widely  different  character.  Thus,  in  the  liver 
and  the  spleen  of  guinea-pigs  coagulation-necrosis  occurs, 
without  true  caseation  ;  in  monkeys  rapid  softening  takes 
place,  with  the  formation  of  a  thin,  diffluent  purulent  secre- 
tion ;  in  the  tuberculosis  of  cows  there  is  simultaneous  cal- 
cification and  caseation.  The  most  common  product  of 
tubercle-bacilli  in  animals  is,  however,  the  tubercle,  which 
is  also  always  encountered  in  connection  with  the  experi- 
mental development  of  tuberculosis,  and  which  resembles 
in  every  respect  the  tuberculous  nodule  of  human  tuber- 
culosis to  be  described  shortly. 

Portals  of  Entry  for  the  Tubercle-bacillus  in  Human 
Beings. — The  tubercle-bacillus  most  frequently  gains  en- 
trance into  the  human  body  through  the  respiration  by  way 
17 


258  CLINICAL  BACTERIOLOGY. 

of  the  lungs.  The  frequency  of  occurrence  of  other  forms 
of  tuberculous  disease  falls  far  behind  that  of  involvement 
of  the  lungs.  The  next  most  important  portal  of  entry  is 
the  digestive  tracts  which  plays  an  important  role  especially 
in  the  etiology  of  tuberculosis  in  early  life.  Under  these 
conditions  the  infection  gives  rise,  on  the  one  hand,  to 
swelling  of  the  cervical  lymph-glands,  often  with  conse- 
quent suppuration  (scrofulosis),  and  on  the  other  hand — 
and  more  frequently  without  primary  involvement  of  the 
intestine — to  caseation  of  the  mesenteric  glands,  or  to 
chronic  peritonitis.  In  adults  the  digestive  tract  is  rela- 
tively seldom  the  portal  of  entrance  for  tubercle-bacilli, 
which  in  that  event  give  rise  to  ulcerative  processes  in  the 
intestine.  Breaches  in  the  continuity  of  the  ski?t  constitute 
a  third  and  not  at  all  uncommon  medium  of  infection  with 
tubercle-bacilli.  Cases  of  cutaneous  tuberculosis  and  of 
tuberculosis  of  wounds  have  in  recent  years  been  reported 
in  considerable  number.  The  so-called  postmortem  tubercle 
must  be  included  in  this  category,  as  careful  investigation 
has  shown.  Further,  the  relationship  of  lupus  to  tubercu- 
losis is  scarcely  any  longer  a  matter  of  serious  doubt. 
Koch  has  succeeded  in  isolating  tubercle-bacilli  in  pure 
culture  from  lupus-nodules. 

The  Pathogenic  Activity  of  the  Bacilli  in  the  Hu- 
man Body. — The  most  characteristic  product  of  tuber- 
cle-bacilli is  the  tubercle.  In  this  the  bacilli  lie  mainly 
within  the  interior  of  the  giant-cells^  partly  at  the  center, 
partly  at  the  periphery  ;  but  the  bacilli  are  to  be  found  also 
in  and  among  the  round  cells  constituting  the  tubercle. 
At  times  the  rods  within  the  giant-cells  appear  not  to  be 
distinctly  stained,  but  disintegrated.  Metschnikofif  considers 
such  appearances  as  the  remains  of  tubercle-bacilli,  and,  in 
consequence,  believes  the  giant-cells  to  be  phagocytes. 
The  specific  nodules  persist  only  for  a  short  time,  and  soon 
disintegrate  into  cheesy-necrotic  material. 

Besides  giving  rise  to  the  formation  of  nodules,  the 
tubercle-bacilli  exhibit  varied  other  pathogenic  activity. 
At  times  they  give  rise  to  serous,  purulent,  or  hemorrhagic 
inflammation,  and  in  rare  cases  even  to  fibrinous  exudation. 
They  may  further  cause  cheesy-necrotic  inflammation 
without  the  previous  formation  of  nodules.  In  some  cases 
the  inflammation  caused  by  the  bacilli  has  from  the  begin- 
ning a  tendency  to   the  formation  of  connective   tissue,  so 


TUBERCULOSIS.  259 

that  it  soon  leads  to  indurative  cicatricial  processes.  This 
great  variability  in  the  anatomic  alterations  induced  by  the 
tubercle-bacilli  explains  the  difference  in  the  point  of  view- 
taken  by  the  pathologic  anatomist  and  by  the  clinician  in 
part  with  regard  to  tuberculosis.  The  anatomist,  in  de- 
scribing the  various  processes,  undertakes  to  separate  them, 
in  spite  of  their  common  etiology,  while  to  the  clinician 
the  etiologic  standpoint  is  alone  productive,  and  therefore 
decisive  ;  and  he  considers  all  diseases  tuberculous  in  whose 
products  tubercle-bacilli  can  be  demonstrated. 

Susceptibility  of  Human  Beings  to  Tuberculosis 
(Predisposition). — Tubercle-bacilli  are  widely  distributed 
throughout  the  inhabited  world.  About  one-seventh  of 
mankind  is  attacked  by  tuberculosis.  The  susceptibility 
of  human  beings  to  the  disease  is,  therefore,  not  especially 
great.  This  view  receives  strong  support  from  the  obser- 
vation that  has  now  been  repeatedly  made  that  the  bron- 
chial glands  of  apparently  healthy  persons  dying  suddenly 
by  accident  contain  living  and  virulent  tubercle-bacilli 
(Loomis,  Pizzini).  In  these  cases  the  bacilli  have  passed 
the  lungs  without  giving  rise  to  disease.  From  the  ana- 
'tomic-bacteriologic  point  of  view  such  individuals  in  perfect 
health  as  harbor  tubercle-bacilli  in  the  bronchial  glands 
or  in  some  other  part  of  their  body  may  be  designated 
tuberculous  ;  while  from  the  clinical  standpoint  only  those 
may  be  considered  tuberculous  that  exhibit  the  clinical 
manifestations  of  tuberculosis. 

From  these  observations  the  significance  of  the  general 
predisposition  to  tuberculosis  must  be  clear.  It  is  evident 
that  only  a  healthy  and  resistant  body  may  harbor  the 
bacilli  without  danger,  whereas  every  debilitating  influence 
facilitates  the  proliferation  of  the  bacilli  within  the  body. 
The  time  of  invasion  by  the  bacilli  and  the  appearance  of 
the  disease  do  not  always  coincide.  Only  a  poorly  nour- 
ished organism  enfeebled  by  grief  and  worry  or  by  pro- 
tracted disease  will  suffer  immediately  after  the  entrance  of 
the  bacilli.  A  special  predisposition  is  conferred  by  the 
abundance  of  sugar  in  the  tissues  in  cases  of  diabetes.  Not 
rarely  contusions  of  the  lung  determine  the  mobilization 
of  previously  latent  tubercle-bacilli  (traumatic  tuberculosis). 
Resistance  to  invasion  by  the  bacilli  appears  to  be 
strengthened  in  human  beings  by  elevated  climates.  Ele- 
vations of  above  2000  meters  (6500  feet)  are  measurably 


260  CLINICAL   BACTERIOLOGY. 

free  from  tuberculosis.  In  the  large  cities  of  Mexico  and 
Pueblo,  situated  between  2000  (6500  feet)  and  2500  meters 
(8200  feet)  above  the  level  of  the  sea,  tuberculosis  is  said 
to  occur  but  rarely,  in  spite  of  the  density  of  the  popula- 
tion. 

Localization  of  Tuberculosis  in  Human  Beings. — In 
human  beings  tuberculosis  usually  remains  localized  to  the 
lungs.  Primary  tuberculosis  of  other  organs  is  rare  in 
adults.  From  the  lungs,  in  the  course  of  the  disease, 
propagation  of  the  bacilli  frequently  takes  place,  partly, 
through  contact,  to  the  pleura  or  other  organs,  partly, 
through  the  intermediation  of  expectorated  or  swallowed 
sputum,  to  the  larynx  and  the  intestine.  In  these  organs  also 
the  tuberculosis  pursues  the  course  of  a  localized  disease. 
The  bacilli  are  carried  by  way  of  the  lymph-paths  and  the 
blood-stream  to  the  membranes  of  the  brain,  the  genito- 
urinary apparatus,  the  bones  and  the  joints,  where  they 
also  give  rise  to  local  disease. 

Highly  febrile  and  rapidly  fatal  general  tuberculosis 
occurs  when  tubercle-bacilli  gain  entrance  into  the  pul- 
monary veins  in  large  number,  and  rapid  dissemination 
through  the  whole  body  follows  (iniliary  tuberculosis^. 
Under  these  conditions  tubercle -bacilli  can  be  found  in  the 
blood. 

Mixed  Infection. — The  symptom-complex  of  tubercu- 
losis in  human  beings  is  at  times  quite  materially  changed 
by  the  presence  and  proliferation  of  other  microorganisms 
in  addition  to  the  specific  exciting  agents.  Especially  in 
pulmonary  cavities  is  this  almost  regularly  the  case.  In 
the  walls  of  such  cavities  sarcinae,  streptococci,  staphy- 
lococci, tetragenus,  the  bacilli  of  blue  pus,  varieties  of 
proteus,  and  others,  may  lodge.  There  thus  result  septic 
and  pyemic  disturbances  that  are  foreign  to  the  tuber- 
culous process  as  such.  Thus,  the  marked  intermittent 
fever  observed  in  so  many  cases  of  tuberculosis  is  proba- 
bly dependent  principally  upon  the  activity  of  streptococci 
(streptococcus-curve). 

Occurrence  and  Distribution  of  Tubercle-bacilli. — The 
tubercle-bacilli  are  found  especially  in  the  lungs  and  in  the 
sputum  in  cases  of  pulmonary  tuberculosis  ;  further,  in  all 
tuberculous  lesions,  including  lupus.  The  blood  contains 
the  bacilli  only  in  cases  of  general  miliary  tuberculosis  and 
then   only  in    small    number.      Infection    may  take    place 


TUBERCULOSIS.  261 

through  all  of  the  products  of  the  disease.  Most  danger- 
ous in  this  respect  naturally  is  tuberculous  sputum.  Of 
less  importance  are  the  feces  in  cases  of  intestinal  tuber- 
culosis, the  urine  in  cases  of  genito-urinary  tuberculosis, 
and  the  pus  in  cases  of  bone-tuberculosis.  By  means  of 
the  sputum  of  tuberculous  patients  who,  instead  of  using 
a  spit-cup,  expectorate  upon  the  floor  or  into  handker- 
chiefs, the  tubercle-bacillus  is  disseminated  in  the  environ- 
ment of  the  patient.  The  microorganism  exhibits  consid- 
erable resistance  to  drying,  and  in  dried  sputum,  for  in- 
stance, retains  its  vitality  and  infectivity  for  six  months.  The 
dried  and  powdered  sputum  is  readily  carried  as  dust  in 
currents  of  air,  and  the  tubercle-bacilli  may  in  this  way 
enter  the  air-passages  of  other  individuals  and  there  give 
rise  to  infection.* 

The  credit  for  pointing  out  this  mode  of  distribution 
for  tubercle-bacilli  belongs  to  Cornet,  who  was  able  to  in- 
fect guinea-pigs  with  tuberculosis  by  means  of  dust  from 
the  walls,  the  floors,  the  furniture,  etc.,  of  hospital -wards 
and  dwelling-apartments  occupied  by  tuberculous  patients 
careless  with  regard  to  the  disposition  of  their  sputum. 
Dust  from  localities  not  invaded  by  tuberculous  patients 
invariably  proved  free  from  tubercle-bacilli. 

Reference  must,  however,  be  made  at  this  place  to  the 
observations  of  Kitasato,  who  showed  by  culture-methods 
that  not  rarely  the  tubercle-bacilli  in  the  sputum  of  tuber- 
culous patients  have  died.  To  what  extent  current  views 
as  to  the  danger  from  tuberculous  sputum  must  be  quali- 
fied by  this  fact  can  not  yet  be  determined  with  certainty. 

A  further  source  of  infection,  whose  importance  must 
not  be  underestimated,  is  constituted  by  the  milk  of  tuber- 
culous cows.  Tuberculosis  in  cattle  is  a  manifestation  of 
the  activity  of  the  tubercle-bacillus — a  tuberculous  process 
that  differs  from  human  tuberculosis  only  in  the  occurrence 
of  calcification  coincidently  with  caseation.  The  milk  of 
cows  suffering  from    tuberculosis  contains  tubercle-bacilli 

*  According  to  observations  made  by  Fliigge,  the  larger  particles  of  sputum, 
both  in  the  fresh  and  in  the  dry  state,  are  less  dangerous.  The  main  danger  is 
believed  to  reside  in  the  fine  particles  of  fluid  that  are  ejected  simultaneously 
with  the  sputum,  and  which  may  float  in  the  air  for  a  long  time  and  be  inhaled 
by  those  with  whom  the  patient  comes  in  contact.  These  small  drops,  as 
Fliigge  has  demonstrated  experimentally,  contain  bacilli.  To  what  extent 
this  fact  should  influence  the  attitude  of  the  physician  with  regard  to  the 
sputum  from  the  prophylactic  standpoint  (p.  266)  can  not  yet  be  defined. 


262  CLINICAL  BACTERIOLOGY. 

with  extraordinary  frequency  (in  fifty  per  cent,  of  diseased 
animals),  even  when  no  tuberculous  changes  are  appreciable 
in  the  udder. 

If  the  frequency  of  tuberculosis  in  cows  is  considered — 
in  some  districts  it  occurs  in  from  twenty  to  fifty  per  cent, 
of  all  the  cows — the  danger  from  the  use  of  unboiled 
or  insufficiently  boiled  milk,  especially  the  mixed  milk  of 
large  cities,  and  particularly  for  children,  must  be  obvious. 
The  hydrochloric-acid  content  of  the  stomach  does  not 
afford  sufficient  protection.  The  tubercle-bacilli  pass  the 
barrier  interposed  by  the  stomach,  and  gain  entrance,  at 
lea^t  in  part,  uninjured,  into  the  intestine:  A  large  pro- 
portion of  the  cases  of  tuberculosis  of  the  intestine  and 
the  peritoneum,  which  is  so  common  in  childhood,  may  be 


^ 


Fig.  56. — Tubercle-bacilli  in  sputum;  Zeiss'  homogeneous  immersion  ^5,  Oc.  4;    X 
about  1000  diameters. 

attributed  to  the  use  of  such  infected  milk.  The  use  of 
meat  from  tuberculous  cows  probably  gives  rise  to  the 
development  of  intestinal  tuberculosis  only  exceptionally. 
The  parts  that  contain  nodules  are  not  permitted  to  be 
offered  for  sale,  and  the  parts  free  from  nodules  contain  no 
bacilli.  Only  in  cases  of  acute  general  miliary  tuberculosis 
could  meat  free  from  tubercles  contain  bacilli,  carried  to  it 
through  the  blood. 

The  diagnostic  demonstration  of  tubercle-bacilli  is  of 
the  highest  importance  for  the  early  recognition  of  tuber- 
culosis. Owing  to  the  specific  behavior  of  tubercle-bacilli 
with  relation  to  stains,  the  demonstration  of  the  micro- 
organisms can  be  made  with  the  greatest  certainty  by  means 
of  the  microscope. 


TUBERCULOSIS.  263 

(a)  Demonstration  of  the  Bacilliin  Sputum  {Pus). — For  con- 
venience of  observation  the  sputum  is  spread  upon  a  black  plate 
or  upon  a  glass  dish  with  a  black  background  (of  paper).  The 
well-known  yellowish  masses  ("lentils")  are  sought  for  and 
one  of  these  or  some  other  purulent  portion  of  the  sputum  is 
placed,  by  means  of  forceps,  upon  a  cover-slip,  and  spread  as 
uniformly  as  possible  upon  its  surface.  After  the  cover-slip  prep- 
aration has  dried  in  the  air,  it  is  passed,  in  the  usual  manner, 
with  the  aid  of  the  forceps,  three  times  through  the  flame  ;  then 
a  few  drops  of  a  freshly  prepared  aniline-water  fuchsin-solution 
or  of  a  carbol-fuchsin  solution  are  added,  and  the  preparation  is 
heated  over  the  flame  until  the  vapor  of  steam  distinctly  escapes. 
After  the  lapse  of  a  minute  the  cover-slip  is  moved  to  and  fro 


F'&'  57- — Tubercle-bacillus  in  sputum  (Fr^nkel  and  Pfeiffer). 

for  several  seconds  in  dilute  nitric  acid  (from  fifteen  to  twenty 
per  cent.),  for  purposes  of  decolorization,  and  it  is  then  intro- 
duced into  alcohol  (seventy  per  cent.)  for  the  removal  of  the 
coloring-matter  dissolved  by  the  nitric  acid.  These  two  man- 
ipulations are  repeated  until  the  preparation  appears  scarcely 
stained.  In  the  preparation  everything  has  now  been  decolor- 
ized, and  only  the  tubercle-bacilli,  if  these  be  present,  have 
retained  the  stain.  The  alcohol  is  removed  with  distilled  water. 
In  order  that  the  stained  tubercle-bacilli  may  be  more  sharply 
differentiated  from  their  surroundings,  the  preparation  is  coun- 
terstained  by  exposure  for  a  short  time  to  the  action  of  a 
dilute  aqueous  solution  of  methylene-blue  or  vesuvin.  It  is  then 
again  rinsed  with  distilled  water,  and  is  finally  mounted  in  the 


264  CLINICAL   BACTERIOLOGY. 

usual  way  for  examination.  The  microscopic  examination  is 
best  made  by  means  of  an  oil-immersion  lens ;  slightly  curved 
bacilli  stained  red  are  looked  for,  and  if  these  are  found,  they 
can  be  tubercle-bacilli  only.  At  times  the  spores  of  mold-fungi 
and  of  bacilli,  portions  of  hair,  fragments  of  horny  epithelial 
cells,  cholesterin-plates,  crystals  of  fatty  acids,  and  similar  ele- 
ments, stained  red,  are  observed,  which  have  resisted  decolor- 
ization ;  but  a  little  experience  will  prevent  confusion  of  these 
with  tubercle-bacilli. 

Decolorizaiion  and  counterstaining  may  be  practised  together 
(B.  Frankel-Gabbet)  by  adding  the  decolorizing  acid  to  the 
solution  employed  in  counterstaining.  After  staining  with 
hot  carbol-fuchsin  solution  the  cover-slip  preparation,  rinsed  in 
water,  is  placed  in  the  following  solution  :  Nitric  acid  20, 
alcohol  30,  water  50,  methylene-blue  to  saturation.  After 
washing  with  water  the  preparation  is  dried  and  mounted  in 
the  usual  manner. 

In  order  to  find  the  tubercle-bacilli  when  these  are  present  in 
small  number,  it  is  advisable,  according  to  the  suggestion  of 
Biedert,  to  dilute  the  sputum  in  a  test-tube  with  water,  to  add 
potassium  or  sodium  hydroxid,  and  to  continue  the  application 
of  heat  until  the  fluid  assumes  a  homogeneous  appearance.  Sed- 
imentation is  then  permitted  to  take  place,  and  the  bacilli,  by 
reason  of  their  weight,  sink  to  the  bottom  of  the  tube.  The 
sediment  is  then  examined  by  one  of  the  methods  just  de- 
scribed. 

{b')  Examination  of  Feces. — A  flake  of  mucus  or  of  pus  is 
selected  from  the  feces,  and  treated  precisely  in  the  manner  de- 
scribed for  sputum. 

{/)  Demonstration  of  Tubercle-bacilli  in  Urine. — The  urine, 
which  is  usually  turbid  from  the  presence  of  pus,  is  permitted 
to  settle  in  a  conical  glass,  or  it  is  centrifugated.  The  sediment 
is  placed  upon  a  cover-slip  in  a  somewhat  thicker  layer  than  the 
sputum,  but  in  other  respects  it  is  treated  in  exactly  the  same 
manner.  In  urine  the  tubercle-bacilli  are  prone  to  lie  together 
in  small  masses  (nests). 

{d  )  Pus  fro7n  cold  abscesses  and  fluid  from  pletiral  effusions 
suspected  to  be  tuberculous  are  often  examined  in  vain  for  tuber- 
cle-bacilli. After  intraperitoneal  injection  of  such  material 
(possibly  after  centrifugation)  guinea-pigs  not  rarely  die  of 
experimental  tuberculosis.  In  this  way  it  is  possible,  although 
after  the  lapse  of  weeks,  to  make  a  diagnosis  of  the  presence  of 
tubercle-bacilli. 

(^)  Staining  of  Tubercle-bacilli  in  Sections. — The  staining  of 
sections  in  hot  solutions  is  not  practicable.  The  preparations 
are,  therefore,  kept  in  the  aniline-water  staining  solution  or  the 
carbolfuchsin  for  from  twelve  to  twenty-five  hours  at  room-tem- 


TUBERCULOSIS.  265 

perature,  or  from  one  or  two  hours  at  37°  C.(98.6°  F.)  in  the 
thermostat.  They  are  decolorized  in  ten  per  cent,  nitric  acid 
for  about  two  minutes  until  they  appear  greenish  blue,  then  in 
seventy  per  cent,  alcohol  until  they  appear  pale  rose.  Next, 
they  are  introduced  into  water,  counterstained  with  a  dilute 
aqueous  solution  of  methylene-blue  or  vesuvin  for  two  or  three 
minutes,  dehydrated  in  absolute  alcohol,  cleared  in  cedar-oil, 
and  mounted  in  Canada  balsam. 

(/)  Examination  of  Milk  for  Tubercle-bacilli. — The  milk  is 
advantageously  first  centrifugated.  Before  being  stained  the 
cover-slip  preparations  are  placed  for  from  four  to  six  minutes 
in  chloroform  for  extraction  of  the  fat.  On  their  removal  the 
chloroform  is  permitted  to  evaporate. 


Fig.  58. — Tubercle-bacilli  in  the  urine;  from  a  case  of  tuberculous  cystitis  (Jakob). 

The  demonstration  of  tubercle-bacilli  in  the  sputum  ren- 
ders unequivocally  certain  the  diagnosis  of  pulmonary 
tuberculosis.  A  negative  result  permits  of  a  definite  con- 
clusion only  when  frequently  repeated.  The  demonstration 
of  tubercle-bacilli  in  the  stools  permits  of  a  diagnosis  of 
intestinal  tuberculosis  only  in  the  absence  of  primary  pul- 
monary tuberculosis.  If  this  is  present,  it  may  be  con- 
cluded that  sputa  have  been  swallowed,  the  bacilli  of 
which  appear  in  the  feces  unchanged.  For  this  reason 
examination  of  the  stools  but  rarely  yields  practical  results. 
If  urinary  sediment  is  stained  from  a  suspicion  of  genito- 
urinary tuberculosis,  the  possibility  of  confusion  with 
smegma-bacilli  (see  Syphilis)  must  be  considered,  as  these 
bacteria  also,  when  exposed  to  acids,  retain  tenaciously  the 


266 


CLINICAL   BACTERIOLOGY. 


stain  taken  up.  Smegma-bacilli  are,  however,  readily  to  be 
distinguished  from  tubercle-baciUi  in  that  they  are  decolor- 
ized within  a  minute  in  absolute  alcohol,  whereas  the 
tubercle-bacilli  retain  their  stain  under  like  conditions. 

Prophylaxis. — The  principal  source  of  tuberculous  in- 
fection is  the  sputum  of  tuberculous  patients.  If  it  were 
possible  to  render  all  sputum  containing  tubercle-bacilli  in- 
nocuous, complete  suppression  of  tuberculosis  might  be 
conceivable.  The  too  zealous  pursuit  of  this  ideal  aim  would, 
however,  lead  to  hardship  on  the  part  of  tuberculous 
patients,  so  that  in  application  the  principle  must  be  modi- 
fied in  accordance  with  humane  considerations.  Besides,  a 
certain  degree  of  moderation  is  permissible  in  this  connec- 
tion, as  the  liability  to  the  disease  may  be  diminished  in  not 
less  degree  by  increasing  the  individual  resistance — that  is, 
by   improving   the    general     conditions    of    life — than    by 


Fig-  59.— Pasteboard  spit-cup  for  receiving  infectious  sputum.    After  being  used,  the 
pasteboard  can  be  removed  from  the  steel  frame  and  burned. 


destruction  of  the  bacilli.  In  any  event,  however,  the  fol- 
lowing principles  should  be  enforced,  or  at  least  impressed 
upon  the  general  public  :  The  tuberculous  patient  should 
expectorate  only  into  a  spit-cup  of  glass  or  porcelain,  filled 
with  water,  in  order  that  there  may  be  no  opportunity  for 
the  sputum  to  dry  and  to  be  converted  into  dust.  In  this 
conversion  into  dust  resides  the  greatest  danger,  and  for 
this  reason  the  addition  to  the  cup  of  all  substances  capa- 
ble of  generating  dust,  such  as  sand,  ashes,  etc.,  should  be 
avoided.  Disinfection  of  the  sputum  can  not  be  accom- 
plished by  means  of  antiseptic  agents,  as  most  of  these  sub- 
stances cause  coagulation  of  the  albuminous  constituents 
of  the  sputum  and  thus  give  rise  to  a  dense  membrane 
that  surrounds  the  mass  and  shields  the  tubercle-bacilli  in 
the  interior  from  contact  with  the  disinfectant.     In  general, 


TUBERCULOSIS.  267 

it  will  suffice  to  empty  the  sputum-cup  into  the  water- 
closet,  as  the  tubercle-bacilli  are  sure  to  die  in  putrid  mix- 
tures. In  hospitals  it  will  be  best  to  sterilize  the  sputa, 
together  with  other  excretions,  by  boiling.  (See  Disinfec- 
tion, Appendix.)  Especial  attention  should  be' paid  to  the 
tuberculous  patient  out  of  doors.  Sputum  expectorated  in- 
discriminately upon  the  street  is  especially  dangerous,  be- 
cause when  reduced  to  powder,  innumerable  persons  are 
exposed  to  the  liability  of  infection.  The  use  of  the  hand- 
kerchief to  receive  the  expectoration  is  likewise  not  without 
risk,  because  here,  also,  there  is  danger  of  drying  and  con- 
version into  dustj.  For  this  reason  it  has  been  wisely  re- 
commended that  tuberculous  patients  carry  with  them  small 
pocket-flasks  for  the  reception  of  the  expectorated  matters 
— for  instance,  Dettweiler's  spit-cup.  It  must,  however,  not 
be  overlooked  that  certain  insurmountable  difficulties  stand 
in  the  way  of  a  general  employment  of  such  flasks,  and  that 
a  careful  use  of  the  handkerchief  may  yet  be  permitted. 

The  rooms  occupied  by  tuberculous  patients  should  be 
cleansed  quite  frequently  with  moist  materials,  in  order  to 
prevent  the  conversion  into  dust  of  the  sputum  that  has 
possibly  become  deposited  on  the  floor  or  on  the  furniture. 
After  the  death  of  a  tuberculous  patient  the  rooms  previ- 
ously occupied  and,  besides,  all  articles  with  which  he  came 
in  contact  should  be  disinfected  with  the  same  care  that  is" 
observed  in  other  infectious  diseases.  In  hospitals,  prisons, 
etc.,  those  suffering  from  tuberculosis  should  be  separated 
from  the  others. 

In  cases  of  intestinal  and  genito-urinary  tuberculosis  and 
of  tuberculous  suppuration  care  should  be  taken  that  the 
feces  or  the  urine  or  the  pus  is  rendered  innocuous. 
Sexual  i7itercourse  should  be  forbidden  those  suffering  from 
genito-urinary  tuberculosis,  as  the  disease  may  be  trans- 
mitted through  the  spermatic  fluid. 

Finally,  it  may  be  stated  that  milk  should  never  be  drunk 
unboiled,  but  should  always  be  boiled  before  being  used. 
It  is  best  to  forbid  the  use  of  meat  from  a  tuberculous  ani- 
mal, or  at  least  to  permit  its  sale  only  when  thoroughly 
cooked,  if  the  disease  is  not  strictly  localized  to  a  single 
organ. 

Heredity  of  Tuberculosis. — A  distinction  is  made 
between  direct  inheritance  of  tuberculosis — the  disease 
'being  transmitted  as   such  to   the  offspring — and  indirect 


268  '  CLINICAL  BACTERIOLOGY. 

inheritance — only  the  tendency  or  the  predisposition  to  the 
disease  being  transmitted.  With  indirect  inheritance  the 
children  of  a  tuberculous  father  or  mother  have  inherited 
a  feeble  body,  a  small  chest — in  brief,  the  tuberculous 
habitus.  They  are  not  tuberculous,  but  are  greatly  predis- 
posed to  tuberculosis  (sujets  tuberculisables,  Peter). 

Cases  of  direct  inheritance — that  is,  of  congenital  tuber- 
culosis— are  known  both  in  human  pathology  and  in  vet- 
erinary medicine,  but  their  number  is  extremely  small  in 
comparison  with  the  wide  distribution  of  the  disease.  The 
same  statement  is  applicable  to  tuberculosis  in  the  first  few 
days  of  life.  On  the  other  hand,  the  mortality  from  tuber- 
culosis in  early  infancy,  especially  in  the  first  year  of  life, 
is  quite  enormous.  Most  observers  assume  that  this  high 
mortality  is  readily  explained  by  the  numerous  sources  of 
infection  to  which  the  child  of  tuberculous  ancestry  is  ex- 
posed after  birth.  The  kiss  of  a  tuberculous  father,  the 
milk  of  a  tuberculous  mother,  are  equally  dangerous,  inde- 
pendently of  the  fact  that  with  neglect  in  the  treatment  of 
the  sputum  on  the  part  of  the  parents  there  is  also  afforded 
frequent  opportunity  for  inhalation  of  the  tubercle-bacilli. 
That  the  disease  is  so  prevalent  and  so  fatal  in  the  first 
year  of  life  is  to  be  explained  by  the  fact  that  at  this  period 
the  children  are  more  predisposed,  and  less  resistant,  to  the 
tubercle-bacillus  than  later.  The  relation  in  these  cases 
would  thus  be  one  of  indirect  inheritance  and  not  one  of 
true  congenital  tuberculosis. 

Baumgarten,  probably  the  most  earnest  advocate  of  the 
doctrine  of  direct  inheritance  of  tuberculosis,  takes  the 
ground  that  the  tubercle-bacilli  are  transmitted  to  the  em- 
bryo during  fetal  life.  The  inherited  germ,  however,  does 
not  proliferate  because  the  tissues  of  the  new-born  child, 
through  their  vital  activity,  offer  considerable  resistance.  The 
bacilli  thus  to  a  certain  degree  remain  latent  in  the  lymph- 
glands,  the  bone-marrow,  etc.,  later,  under  the  favoring 
influence  of  traumatism,  intercurrent  disease,  or  diminution 
in  the  vital  energy  of  the  cells,  to  unfold  their  deleterious 
activity.  This  view  is  supported  by  the  observations  of 
Landouzy,  Martin,  Birch-Hirschfeld,  Schmorl,  and  others, 
who  found  tubercle-bacilli  in  apparently  healthy  organs 
from  fetuses  born  of  tuberculous  mothers.  On  the  other 
hand,  Gartner  lays  emphasis  upon  the  fact  that  the  infantile 
cells  do  not  exert  an   especially  injurious   influence   upon 


TUBERCULOSIS.  269 

the  bacilli.  Tuberculosis  pursues  a  more  rapid  course  in 
youth  than  at  a  later  age,  and  experimentally  no  difference 
can  be  observed  between  young  and  old  animals.  On  the 
other  hand,  Gartner  is  of  the  opinion  that  in  the  act  of  par- 
turition, perhaps  in  consequence  of  tears  in  the  placenta, 
the  bacilli  pass  over  from  the  mother  to  the  child.  The 
possibility  of  such  a  transfer  of  the  tubercle-bacilli  to  the 
embryo  Gartner  has  himself  demonstrated  with  certainty 
in  his  experiments  upon  mice,  canary-birds,  and  rabbits. 
Fetal  infection,  according  to  this  view,  takes  place  late,  and 
in  all  probability  it  is  effected  through  only  one  or  a  few 
bacilli.  Consequently,  the  disease  can  not  be  manifest  at 
birth,  but  it  becomes  evident  only  after  the  child  has  lived 
for  some  time  outside  the  uterus.  Also,  the  cases  of  pri- 
mary tuberculosis  of  the  skin,  the  joints,  the  bones,  the 
glands,  the  spleen,  the  liver,  which  are  not  uncommon  in 
early  childhood,  indicate  a  hematogenous  fetal  infection. 
The  objection  that  the  frequency  of  pulmonary  tuberculosis 
is  opposed  to  the  preponderant  occurrence  of  hematog- 
enous fetal  transmission,  is,  according  to  Gartner,  justified ; 
but  it  is  to  be  borne  in  mind  that,  in  spite  of  exhibiting  the 
most  conspicuous  changes,  the  lungs  are  by  no  means 
always  the  seat  of  the  primary  tuberculous  lesion.  The 
lungs  of  human  beings  are,  *'by  reason  of  their  constitu- 
tion, their  situation,  and  their  chemistry,  especially  adapted 
for  the  lodgment  and  development  of  tubercle-bacilli," 
which  may  gain  entrance  from  any  primary  focus  (perhaps 
of  fetal  origin).  The  question  whether  tuberculosis  may 
be  transmitted  to  the  embryo  from  the  father  Gartner  an- 
swers in  the  negative.  He  rendered  male  rabbits  and 
guinea-pigs  tuberculous  by  injection  of  bacilli  into  the  tes- 
ticles, and  he  then  brought  them  in  contact  with  females, 
but  the  offspring  were  not  tuberculous  ;  whereas  when  the 
bacilli  were  present  in  the  seminal  fluid  in  considerable 
numbers,  the  females  were  infected. 

Therapeutic  Experiments. — Many  efforts  have  been 
made  to  confer  immunity  to  tuberculosis,  and  to  effect  the 
cure  of  tuberculosis  by  the  same  means.  This  end  was 
sought  by  the  use  of  the  blood-scrum  of  animals  that  are 
relatively  refractory  to  the  disease,  such  as  dogs  (Richet  and 
Hericourt)  and  goats.  Further,  attempts  have  been  made 
to  establish  immunity  by  means  of  attenuated  or  sterilized 
cultures  of  tubercle-bacilli.     All  of  these  endeavors,  how- 


270  CLINICAL  BACTERIOLOGY. 

ever,  have  thus  far  proved  unsuccessful.  Richet  maintained 
for  a  time  that  he  had  succeeded  in  vaccinating  dogs  by 
inoculating  them  with  the  bacilli  of  fowl-tuberculosis,  or 
with  small  amounts  of  mammalian  tubercle-bacilli,  but  his 
observations  have  not  been  confirmed. 

R.  Koch  endeavored  to  secure  a  direct  and  specific  in- 
fluence upon  the  tuberculous  lesions  by  means  of  tuberculiji. 
This  product  was  obtained  by  concentrating  mature 
bouillon-cultures  of  tubercle-bacilli  (from  six  to  eight 
weeks  old)  to  one-tenth  of  their  volume  over  the  water- 
bath,  and  freeing  the  culture-fluid  of  the  bodies  of  the 
dead  bacilli  by  means  of  filtration  through  porcelain  or 
gravel.  Experimental  observation  showed  that  healthy 
guinea-pigs  withstand  without  harm  subcutaneous  injec- 
tions of  as  much  as  two  cubic  centimeters  of  tuberculin, 
whereas  tuberculous  animals,  infected  four  weeks  previ- 
ously, succumb  to  a  dose  of  0.6  cu.  cm.  On  autopsy 
in  cases  of  animals  dead  after  injections  of  tuberculin  the 
following  conditions  were  found  :  The  site  of  tuberculous 
inoculation  was  markedly  reddened,  as  well  as  the  adjacent 
lymph-glands.  In  the  viscera  enormous  dilatation  of  the 
capillaries  surrounding  the  tuberculous  foci  was  discernible 
microscopically.  The  hemorrhage-like  spots  on  the  sur- 
face of  the  liver  were  quite  pathognomonic.  In  tuber- 
culous guinea-pigs  treated  with  small  doses  of  tuberculin 
(at  first  I  mg.,  increasing  to  o.  i  and  0.2  gram)  a  striking 
effect  upon  the  morbid  condition  was  observed.  Improve- 
ment in  the  primary  area  of  inoculation  in  the  abdominal 
wall  was  observed,  with  diminution  in  size  of  the  adjacent 
tumid  lymph-glands,  but  complete  recovery,  as  later  reports 
from  the  Koch  Institute  have  shown,  occurred  only  excep- 
tionally. The  animals  survived  for  a  longer  time  than 
tuberculous  control  guinea-pigs  not  treated  with  tuberculin. 
Postmortem  examination  in  the  case  of  animals  treated 
disclosed  marked  retrogression,  in  part  cicatrization  of  the 
tuberculous  lesions  in  the  abdominal  viscera,  but  eventually 
death  resulted  from  pulmonary  tuberculosis. 

The  curative  influence  of  tuberculin  is  explained  by  Koch 
on  the  assumption  that  as  a  result  of  its  action  the  necrotic 
substance,  which  is  always  present  in  the  neighborhood  of 
a  tuberculous  lesion,  is  increased  in  amount,  so  that  coag- 
ulation-necrosis occurs  throughout  a  wide  extent,  and 
this   hinders    the  bacillus    in    its    further   growth,   and  at 


TUBERCULOSIS.  271 

times  causes  its  death.  Opposed  to  the  original  opinion  of 
Koch  is  the  fact  that  the  action  of  tuberculin  upon  tuber- 
culous tissues  is,  however,  not  specific,  and  not  confined  to 
it  alone.  As  Romer  was  the  first  to  show,  the  products 
derived  from  other  microorganisms  by  boiling  (proteins) 
induce  precisely  the  same  reaction. 

The  influence  of  tuberculin  upon  healthy  and  diseased 
human  beings  has  been  tested  from  both  a  diagnostic 
and  a  therapeutic  standpoint  in  so  large  a  number  of  cases 
that  a  definite  opinion  can  now  be  expressed.  Diagnos- 
tically,  tuberculin  has  proved  an  exceedingly  delicate 
reagent  for  the  detection  of  tuberculous  lesions.  Subcu- 
taneous injection  of  five  milligrams  is  followed  by  no 
appreciable  effect  in  healthy  persons.  In  tuberculous  in- 
dividuals, however,  this  dose  gives  rise  to  fever  of  moderate 
degree,  lasting  for  several  hours,  while  at  the  same  time  a 
local  reaction,  manifested  by  redness  and  swelling,  takes 
place  in  the  tuberculous  lesions  in  so  far  as  these  are  acces- 
sible to  examination.  Initial  doses  of  ten  milligrams  cause 
in  tuberculous  subjects  greater  and  more  protracted  eleva- 
tion of  temperature,  together  with  headache,  nausea,  and' 
vomiting.  Similar  doses  cause  also  in  healthy  persons 
febrile  disturbance  and  general  manifestations. 

The  undoubted  diagnostic  titility  of  minimal  doses  of 
tuberculin  is  rendered  practically  unavailable  from  the  fact 
that  the  injection  is  followed  by  febrile  movement  in  all 
persons  who  harbor  tubercle-bacilli,  even  when  these  are 
latent  and  encapsulated — as,  for  instance,  in  lymphatic  or 
bronchial  glands.  It  has  been  found,  in  numerous  cases, 
that  apparently  healthy  persons  develop  fever  after  injec- 
tions of  tuberculin  in  the  same  way  as  tuberculous  patients. 
From  what  has  been  said  with  regard  to  the  frequent 
occurrence  of  tubercle-bacilli  in  healthy  persons,  this  mani- 
festation should  not  occasion  surprise  ;  but  a  diagnosis  of 
tuberculosis  is  not,  in  a  clinical  sense,  justifiable  when 
merely  the  presence  of  tubercle-bacilli  within  the  organism 
has  been  demonstrated.  The  conditions  are  quite  different 
in  the  diagnosis  of  tuberculosis  than  in  that  of  diphtheria 
or  of  cholera.  It  is  of  the  greatest  sanitary  importance  to 
recognize  the  existence  of  diphtheria  or  of  cholera  in  per- 
sons in  whom,  though  apparently  in  perfect  health,  the 
exciting  agents  of  those  diseases  are  present  in  the  saliva 
or  in  the  dejections,  because  further  cases  of  infection  may 


272  CLINICAL  BACTERIOLOGY. 

arise  through  them.  In  the  case  of  tuberculosis,  however, 
only  those  individuals  are  dangerous  who  present  actual 
symptoms  of  disease— that  is,  those  who  throw  off  tubercle- 
bacilli  with  the  sputum  or  other  excretions.  In  the  case  of 
tuberculosis  thus  the  diagnostic  requirements  on  the  part 
of  both  physician  and  sanitarian  are  identical  ;  whereas  in 
the  case  of  cholera  and  of  diphtheria  these  diverge.  From 
the  foregoing  considerations  the  employment  of  injections 
of  tuberculin  for  diagnostic  purposes  should  be  restricted  to 
cases  of  actual  disease  of  obscure  etiology.  Frequently,  how- 
ever, under  such  conditions  also  this  test  has  been  abstained 
from  because  experience  in  some  cases  has  demonstrated 
the  possibility  of  the  bacilli  being  disseminated  from  a  pre- 
viously circumscribed  focus  throughout  the  entire  body  as 
the  result  of  an  injection  of  tuberculin. 

The  tJierapeutic  employment  of  tuberculin  in  tuberculous 
individuals  has  yielded  the  following  results  :  Lupus  is  at 
times  favorably  influenced  by  tuberculin.  Extensive  areas 
of  lupus  undergo  necrosis  and  are  exfoliated,  so  that  com- 
plete recovery  may  take  place ;  but  no  case  is  yet  known 
in  which  recurrence  has  not  taken  place.  Ulcers  of  the 
larynx  clear  in  a  most  remarkable  manner  after  injections 
of  tuberculin,  and  undergo  healing  ;  but  in  these  cases  also 
recurrence  generally  takes  place.  Intestinal  and  peritoneal 
tuberculosis  appears  to  pursue  a  relatively  favorable  course 
when  treated  with  injections  of  tuberculin.  Under  all  of 
these  conditions,  however,  the  fact  stands  out  that  the 
results  are  not  final,  because  tuberculin  lacks  immunizing 
properties.  Tuberculous  disease  of  bones  and  joints  is  not 
at  all  influenced  by  the  treatment.  With  relation  to  pul- 
monary tuberculosis,  it  may  be  stated  with  certainty  that  in 
the  presence  of  advanced  infiltration,  of  cavity-formation, 
and  of  mixed  infection  success  can  not  be  attained  with 
tuberculin-treatment ;  and  it  is  questionable,  further,  whether 
even  incipient  tuberculosis  can  be  cured  by  the  original 
method  of  Koch.  A  large  number  of  unfavorable  results 
are  opposed  to  a  small  number  of  undoubted  cases  of  in- 
cipient tuberculosis  treated  with  favorable  results.  The 
objection  may  be  raised  that  the  results  obtained  in  these 
cases  are  not  attributable  to  the  specific  agent,  but  to  the 
nutritive  and  general  therapeutic  measures  employed  simul- 
taneously. Tuberculin  has  been  almost  entirely  abandoned 
by  physicians.    In  veterinary  medicine,  especially  in  France, 


TUBERCULOSIS.  273 

it  is  still  employed  on  a  large  scale  for  diagnostic  purposes, 
and,  it  appears,  with  great  success. 

The  New  Tuberculin-preparations,  TO  and  TR. — After 
the  failure  of  the  original  tuberculin,  Koch  applied  himself 
unremittingly  to  the  improvement  of  tuberculin-prepara- 
tions. He  found  that  immunity  could  not  be  conferred 
upon  animals  by  subcutaneous  injection  of  unchanged 
tubercle-bacilli.  He,  therefore,  undertook  to  disintegrate 
the  tubercle-bacilli  mechanically,  in  order  to  render  them 
the  more  easily  absorbable.  The  cultures  were  dried  in  a 
vacuum  and  rubbed  up  in  an  agate  mortar  without  addition 
until  only  a  small  number  of  bacilli  were  visible  micro- 
scopically. The  powder  was  mixed  with  distilled  water,. 
and  centrifugated  for  from  a  half  to  three-quarters  of  an 
hour.  In  this  way  Koch  effected  a  separation  into  two 
layers — an  upper  transparent,  opalescent  layer,  which  con- 
tained no  bacilli,  and  a  densely  adherent  lower  layer.  The 
latter  was  again  dried,  rubbed  up,  and  centrifugated  in  the 
manner  described,  and  the  entire  process  was  repeated  until 
all  the  bacilli  had  practically  been  reduced  to  solution. 
Koch  then  determined  that  the  solutions  thus  obtained 
were  readily  absorbable,  and  that  they  did  not  give  rise  to 
the  formation  of  abscesses.  It  was  found,  however,  that 
the  fluid  obtained  from  the  first  centrifugation  exhibited 
reactions  different  from  that  obtained  from  the  second  and 
from  subsequent  centrifugations.  Koch  designated  the  first 
tuberculin  O  {obere,  upper — TO)  ;  and  all  of  the  others, 
which  were  alike  in  their  reaction,  tuberculin  R  (residue — 
TR).  TO  contains  the  constituents  of  the  tubercle-bacilli 
soluble,  and  TR  those  insoluble,  in  glycerin.  It  can  be 
readily  understood  that  the  properties  of  TO  are  compar- 
able, on  the  whole,  with  those  of  the  original  tuberculin. 
TR,  however,  according  to  Koch,  exhibits  distinct  immuniz- 
ing properties,  and  gives  rise  to  reaction  in  tuberculous 
subjects  only  when  used  in  large  doses.  Its  action  is 
entirely  independent  of  the  reactions  that  played  so  impor- 
tant a  part  when  the  original  tuberculin  was  used.  Koch 
states  that  in  conferring  immunity  with  TR  the  reactions 
may  be  entirely  avoided,  and,  by  carefully  increasing 
the  dose,  tuberculous  subjects  may  be  habituated  quite 
rapidly,  without  any  reaction,  to  considerable  amounts  of 
the  new  remedy.  When  this  has  been  accomplished,  the 
organism  may  be  considered  as  immune  to  the  original 
i8 


274  CLINICAL  BACTERIOLOGY. 

tuberculin  and  to  TO — that  is,  to  all  of  the  bodily  con- 
stituents of  the  tubercle-bacilli. 

The  new  preparations  are  put  upon  the  market  by  the 
Hochst  Works.  For  purposes  of  preservation  20  per  cent, 
of  glycerin  is  added.  One  cubic  centimeter  of  the  new 
tuberculin  TR  contains  10  mg.  of  solid  substance.  After 
appropriate  dilution  with  sterile,  physiologic  solution  of 
sodium  chlorid  (if  the  daughter-solution  is  to  be  preserved 
for  some,  time,  20  per  cent,  of  glycerin  is  added),  the  treat- 
ment is  begun  by  injecting  -^-^  mg.  The  next  higher 
dose  is  to  be  administered  on  the  second  day,  and  it  should 
be  of  such  an  amount  that  temperature -elevations  of  more 
than  ^°  do  not  take  place.  Should  these  occur,  the  next 
injection  must  be  deferred  until  the  temperature  has  again 
become  normal.  As  a  rule,  the  treatment  is  suspended 
when  the  dose  reaches  20  mg.,  and,  if  no  reaction  follows, 
the  same  amount  is  repeated  at  considerable  intervals. 

If  it  is  desired  to  immunize  healthy  animals,  as  large  a 
quantity  is  injected  at  first  as  is  well  borne  by  them — for 
instance,  in  the  case  of  guinea-pigs,  2  or  3  mg.  In  this 
way  Koch  was  able  to  immunize  a  considerable  number  of 
guinea-pigs  to  highly  virulent  tubercle- bacilli.  The  height 
of  the  immunity  is  attained  two  or  three  weeks  after  ad- 
ministration of  the  large  dose.  From  this  it  appears  that 
in  therapeutic  experiments  on  artificially  infected  guinea- 
pigs,  which  rapidly  succumb  to  the  disease,  the  treatment 
must  be  instituted  quite  early — not  later  than  two  weeks 
after  the  introduction  of  the  virus. 

Koch  recommends  that  this  new  remedy  be  employed 
only  in  recent,  pure  cases  of  tuberculosis,  uncomplicated 
by  mixed  infection.  Patients  who  exhibit  a  temperature 
above  38°  C.  (100.4°  F.)  are  not  adapted  to  the  new 
method  of  treatment.  In  cases  of  lupus  Koch  obtained 
considerable  improvement  without  noteworthy  local  re- 
action. At  the  conclusion  of  his  communication  Koch 
emphasizes  the  fact  that  perhaps  combinations  of  TO  and 
TR  with  serum-preparations  made  from  TO  and  TR  may 
more  quickly  lead  to  the  desired  results.  The  clinical 
reports  that  have  thus  far  been  made  with  regard  to  the 
treatment  of  tuberculosis  with  TR  show  the  freedom  from 
danger  that  attends  the  use  of  the  remedy,  but  little  as  to 
its  therapeutic  utility.  Personally,  no  noteworthy  results 
have  been  obtained  in  fifteen  cases  of  pulmonaiy  tubercu- 


FOWL-TUBERCULOSIS.  275 

losis  thus  treated.  Behring  is  of  the  opinion  that  tuber- 
cuHn  R  is  less  well  adapted  for  therapeutic  employment  in 
the  case  of  human  beings  than  for  the  fundamental  inocu- 
lation of  animals  for  the  purpose  of  further  immunization. 
His  own  experiments,  in  connection  with  von  Lingelsheim, 
in  the  preparation  of  a  serum  for  tuberculosis,  according  to 
his  statement  read  before  the  Fifteenth  Congress  for  In- 
ternal Medicine,  appear  promising.  Behring  and  von  Lin- 
gelsheim in  their  experiments  employed  dry,  highly  viru- 
lent, pure  cultures  of  tubercle-bacilli.  According  to  Beh- 
ring, the  tubercle-bacilli  contain  various  substances,  but 
only  one  of  the  tuberculosis-toxins  appears  to  possess  im- 
munizing properties.  With  the  aid  of  this  toxin  it  may  be 
hoped  that  a  curative  serum  or  an  antitoxin  will  be  pro- 
duced, as  in  the  case  of  diphtheria  and  of  tetanus  ;  but  ac- 
cording to  Behring,  years  may  elapse  before  this  serum  will 
prove  sufficiently  powerful  to  be  introduced  into  general 
practice. 

FOWL-TUBERCULOSIS. 

The  bacillus  of  fowl-tuberculosis  is  closely  related  to  the 
bacillus  of  human  and  mammalian  tuberculosis.  It  is  somewhat 
longer  and  thinner,  and  exhibits  more  frequently  bulbous  and 
branched  variations.  It  is  more  easily  stained,  but  it  retains  the 
stain  with  similar  tenacity.  It  is  not  so  fastidious  with  regard  to 
culture-media,  and  it  develops  upon  ordinary  agar  and  upon  or- 
dinary bouillon.  The  addition  of  glycerin,  however,  materially 
favors  its  growth,  which,  on  the  whole,  is  more  rapid  than  that 
of  the  bacillus  of  mammalian  tuberculosis.  The  cultures  are 
not  so  dry,  but  more  moist,  and  on  solid  media  they  form  a 
coherent  coating  that  bridges  over  the  water  of  condensation. 
All  cultures  constantly  exhibit  a  yellowish  discoloration.  At 
temperatures  of  42°  C.  (107.6°  F.),  43°  C  (109.4°  F.)  or  45° 
C.  (113°  F. )  the  bacilli  of  fowl-tuberculosis  thrive  as  luxur- 
iantly as  at  a  temperature  of  37°  C.  (98.6°  F.).  This  is  their 
most  radical  distinguishing  feature  as  compared  with  the  bacilli  of 
human  tuberculosis,  which  will  not  develop  at  this  temperature. 
If  the  two  varieties  are  considered  identical,  this  difference 
must  be  explained  by  the  fact  that  by  reason  of  their  residence 
in  the  body  of  birds,  which  naturally  have  a  higher  temperature 
(41°  0.-105.8°  F.,  or  42°  €.  —  107.6°  F.),  the  bacilh  have 
adapted  themselves  to  a  higher  temperature.  The  bacilli  of 
fowl-tuberculosis  are  even  more  resistant  to  heat  than  those  of 
human  tuberculosis,  being  destroyed  by  exposure  for  fifteen 
minutes  to  a  temperature  of  70°  C.  (158°  F.). 


276  CLINICAL   BACTERIOLOGY. 

Occurrence  of  the  Bacilli. — The  bacilli  are  present  in 
the  tuberculous  lesions  of  fowl,  which  consist  of  dense, 
tumor-like  masses  containing  calcareous  deposits.  Giant- 
cells  are  present  in  but  small  number.  Fowl-tuberculosis 
has  been  observed  in  rare  cases  also  in  human  beings  and 
in  mammals. 

Experimental  Development  of  Fowl- tuberculosis. — 
Most  birds  are  highly  susceptible,  and  infection  may  take 
place  through  all  portals  of  entry.  According  to  Baum- 
garten  fowl-tuberculosis  occurring  spontaneously  is  con- 
genital in  almost  all  instances.  Rabbits,  also,  succumb  to 
infection  with  fowl-tuberculosis.  Guinea-pigs  and  dogs 
prove  rather  refractory,  without,  however,  being  entirely 
immune.  On  the  whole,  the  bacilli  of  fowl-tuberculosis 
thrive  poorly  in  mammals,  and  mammalian  tuberculosis, 
conversely,  develops  only  with  difficulty  in  birds. 

Diagnosis. — The  examination  of  tuberculous  masses  for 
bacilli  is  conducted  in  precisely  the  same  manner  as  in  the 
case  of  human  tuberculosis. 


PSEUDaTUBERCULOSIS. 

'^y  pseudo-tuberculosis  is  understood  certain  pathologic 
processes  that  exhibit  the  external  appearance  of  tubercles, 
but  are  dependent  upon  other  causes  than  the  tubercle- 
bacillus.  The  etiology  of  pseudo-tuberculosis  is  quite 
varied.     Among  the  causative  factors  may  be  mentioned  : 

1.  Inanimate  foreign  bodies. 

2.  Animal  parasites. 

3.  Bacteria. 

4.  More  highly  organized  vegetable  parasites. 

The  tuberculosis  due  to  foreign  bodies  may  be  developed 
experimentally  with  ease  through  the  agency  of  all  possible 
substances.  It  is,  however,  not  transmissible  from  animal 
to  animal. 

Pseudo-tiiberculosis  due  to  auimal  parasites  is  observed 
almost  exclusively  in  animals.  Among  the  varieties  that 
are  well  known  is  the  tuberculosis  of  the  cat,  caused  by 
ollulanus  tricuspis  ;  that  of  the  sheep,  due  to  pseudalius 
ovis  pulmonalis  ;  that  of  the  calf,  due  to  strongylus  ruf- 
escens ;  that  of  the  dog,  due  to  strongylus  vasorum. 
Miura  alone  has  observed  a  single  case  in  a  human  being. 


PSEUDO-TUBERCULOSIS.  277 

This  occurred  in  a  man  dying  of  beri-beri,  in  whose  omen- 
tum were  found  fibrous  tubercles  caused  by  the  ova  of  dis- 
toma. 

Bacterial  pseudo-tuberculosis  has  been  often  described  in 
animals.  First  designated  zooglear  tuberculosis  by  Malas- 
sez  and  Vignal,  this  disorder  has  since  been  studied  by 
numerous  observers,  and  most  carefully  by  Preisz.  The 
cause  of  this  affection  consists  in  thick,  short  rods,  fre- 
quently resembling  cocci,  and  forming  filaments.  Spores 
do  not  develop,  and  Gram's  method  fails  to  stain.  Upon 
gelatin-plates  colonies  form  resembling  those  of  the 
typhoid-bacillus,  but  not  causing  liquefaction.     Upon  gela- 


.J .  •..> 

Fig.  60. — Bacillus  pseudo-tuberculosis  from  agar-agar;  X  1000  (Itzerott  and 
Niemann). 

tin  stab-cultures  a  flat,  nail-like  growth  takes  place  ;  upon 
agar  a  grayish,  fetid  coating  forms,  and  upon  potatoes  a 
yellowish  deposit.  In  bouillon  there  is  at  first  flocculent 
turbidity,  then  the  formation  of  a  precipitate  and  a  clear- 
ing up  of  the  supernatant  fluid.  The  bacillus  pseudo- 
tuberculosis is  pathogenic  for  all  rodents  ;  in  slighter  degree, 
also,  for  dogs  and  horses.  The  postmortem  findings  are 
strongly  suggestive  of  true  tuberculosis,  particularly  in  the 
abdominal  organs,  which  are  especially  attacked  by  pseudo- 
tuberculosis. The  differential  diagnosis  is  made  with  great 
ease  The  readiness  with  which  the  bacilli  stain  and  their 
rapidity  of  growth  permit  of  a  decision  without  difficulty. 


278  CLINICAL  BACTERIOLOGY. 

Pseudo-tuberculosis  due  to  more  highly  organized  vegetable 
parasites  likewise  occurs  preferably  in  animals.  Various 
kinds  of  streptothrices  and  aspergilli  act  as  the  etiologic 
factors  under  these  conditions,  particularly  aspergillus 
glaucus  and  fumigatus.  Pigeons  frequently  succumb  to  a 
form  of  miliary  pseudo-tuberculosis,  in  which  the  aspergil- 
lus fumigatus  is  found  inclosed  within  giant-cells  in  the  in- 
terior of  the  granulations.  In  individuals  engaged  in  the 
fattening  of  pigeons  pulmonary  affections  have  been  ob- 
served apparently  dependent  upon  the  same  microbes  ;  at 
least  the  aspergillus  fumigatus  has  been  found  in  the  spu- 
tum of  such  patients.  It  is  assumed  that  the  parasite 
adheres  to  the  grain  used  in  feeding  the  pigeons. 

Eppinger  has  observed  in,  and  cultivated  from,  a  case 
of  pseudo-tuberculosis    a  variety  of  streptothrix  that   he 
designates  cladothrix  asteroides.     With  pure  cultures  of 
this  organism  he  succeeded  in  inducing  the  same  disease  in. 
animals.     (See  Actinomycosis.) 


LEPROSY* 

The  bacillus  of  leprosy  was  discovered  by  Armauer 
Hansen.  It  has  thus  far  not  been  successfully  cultivated. 
In  morphologic  appearance  the  bacilli  closely  resemble 
tubercle-bacilli.  Often,  they  appear  somew^hat  shorter, 
and,  like  these,  they  are  nonmotile.  With  regard  to  tingi- 
bility,  the  bacilli  of  leprosy  occupy  a  position  midway  be- 
tween tubercle-bacilli  and  other  bacteria.  As  a  rule,  they 
prove  susceptible  only  to  the  methods  of  staining  that  are 
applicable  to  tubercle-bacilli.  They  stain  and  decolorize 
somewhat  more  readily,  however,  than  tubercle-bacilli,  and 
they  can  be  stained  also  by  simple  aqueous  solutions 
of  aniline  dyes,  especially  violet  and  fuchsin,  at  room- 
temperature  (Baumgarten).  They  can  be  stained,  further, 
by  Gram's  method. 

In  the  leprous  new-formations  (leprous  nodules)  the 
leprosy-bacilli  lie  mainly  within  the  tissue-cells,  in  the  so- 
called  leprosy-cells.  They  have  been  found  in  the  blood 
only  during  the  febrile  periods. 

E.  Levy  has  cultivated  from  a  case  of  leprosy  a  bacte- 
rium that  in  glycerin-agar  cultures  bears  some  resemblance 
to  the  bacillus  of  mammalian  tuberculosis,  but  that  after 


LEPROSY.  279 

Staining  proves  resistant  to  neither  acid  nor  alcohol.  Micro- 
scopically, it  bears  a  distinct  resemblance  to  the  anaerobic 
actinomyces.  It  presents  bulbous  enlargements  and  rami- 
fications. 

Distribution  of  Leprosy. — Leprosy,  which  formerly 
was  indigenous  to  all  of  Europe,  is  now  restricted  to  Nor- 
way, Livonia,  Turkey,  the  Crimea,  and  Southern  Italy. 
Of  late,  isolated  cases  of  the  disease  have  again  been 
encountered  in  Eastern  Prussia.      In  countries  outside  of 


^P^.IS? 


Fig.  6i. — Bacilli  shown  in  a  section  of  the  tongue  in  a  case  of  tubercular  leprosy  ; 
X  600.  The  bacilli  ar«  extracellular:  B,  Bacilli  in  groups;  Z,  Z,  zooglear  masses, 
large  rounded  masses  of  bacilli ;  C,  bacilli  in  chains  (Leloir). 


Europe  leprosy  is  quite  common.  The  small  number  of 
cases  that  are  observed  in  other  places  may  be  explained 
by  importation  from  leprous  localities.  The  disappearance 
of  leprosy  is  undoubtedly  to  be  attributed  to  the  isolation 
— formerly  practised  with  barbaric  severity — of  those 
affected  in  special  leprosy-houses  (leproseries).  From  this 
disappearance  of  the  disease  it  may  be  concluded  that  it  is 
contagious,  but  this  has  not  been  demonstrated  with  cer- 
tainty, and  is  doubted  by  some  physicians. 

Experiments  on  Animals. — The  experimental  develop- 


280  CLINICAL  BACTERIOLOGY. 

ment  of  leprosy  in  animals  has  thus  far  not  succeeded. 
The  only  observations  that  can  be  considered  as  positive 
are  those  of  Melcher  and  Ortmann,  in  which,  after  inocu- 
lation of  the  anterior  chamber  of  the  eye,  general  infection 
developed  in  rabbits  ;  but  these  are  interpreted  by  most 
observers  as  indicating  that  in  the  case  or  cases  from 
which  the  infecting  material  was  obtained  there  existed 
mixed  infection  with  leprosy  and  tuberculosis,  and  that  the 
inoculated  animals  died  of  tuberculosis.  All  of  the  attempts 
at  inoculation  of  human  beings  have  proved  equally  unsuc- 
cessful, or  at  least  not  above  criticism.  Danielsen,  who  inoc- 
ulated himself  and  others  with  nodular  masses,  blood,  etc., 
from  leprous  patients,  obtained  completely  negative  results. 
On  the  other  hand,  the  experiments  of  Arning  terminated 
positively  ;  this  observer  succeeded  in  transmitting  leprosy 
to  a  prisoner  condemned  to  death.  This  case  has,  how- 
ever, been  seriously  doubted,  as  the  period  of  incubation 
was  strikingly  short,  being  only  sixteen  months,  whereas 
the  whole  course  of  the  disease  covered  only  five  years,  and 
as,  besides,  the  inoculated  person  was  a  member  of  a  race 
highly  susceptible  to  leprosy,  and  cases  of  leprosy  had 
already  occurred  in  his  family. 

Bacteriologic  Diagnosis. — Cover-slip  preparations  are 
made  from  the  contents  and  the  tissue-juice  of  leprous 
nodules,  and  are  stained  in  precisely  the  same  way  as  tuber- 
cle-bacilli. Great  care  must  be  taken  in  the  process  of 
decolorization.  The  sections  of  leprous  tissue  are  not  per- 
mitted to  remain  for  so  long  a  time  in  the  staining  solution 
as  sections  of  tissue  containing  tubercle-bacilli ;  half  an  hour 
is  quite  sufficient. 

In  most  cases  of  leprosy,  as  R.  Koch  was  the  first  to  ob- 
serve, the  nasal  mucus  contains  leprosy-bacilli.  These  are 
derived  from  areas  of  ulcerated  or  swollen  mucous  mem- 
brane on  the  cartilaginous  portion  of  the  nasal  septum. 
The  bacilli  may,  however,  be  present  when  such  visible 
changes  are  absent. 

Heredity  of  Leprosy. — According  to  Baumgarten,  lep- 
rosy, like  tuberculosis,  is  transmissible  by  inheritance.  If 
this  opinion  is  correct,  a  long  period  of  latency  must  be  as- 
sumed for  the  inherited  leprosy -bacilli,  as  the  disease  never 
appears  before  the  second  or  the  third  year  of  life. 

Prophylaxis. — The  best  prophylaxis  unquestionably 
consists  in  isolation  of  leprous  patients  in  special  hospitals. 


INFLUENZA. 


INFLUENZA, 

On  the  first  occurrence  of  influenza  in  the  winter  of 
1889—90  the  numerous  bacteriologic  investigations  under- 
taken failed  to  yield  conclusive  results.  In  the  secretions 
of  influenza-patients  the  ordinary  exciting  agents  only  of 
inflammation,  especially  streptococci  and  lanceolate  diplo- 
cocci,  were  encountered.  Of  the  latter  it  was  stated  that  in 
appearance  and  growth  they  presented  certain  points  of 
distinction  as  compared  with  ordinary  pneumococci.  A 
specific  bacterium  was,  however,  not  found.  In  subsequent 
epidemics  (189 1  and  1892)  Pfeiffer,  in  the  Hygienic  Institute 
at  Berlin,  recognized  a  special  bacillus  as  the  exciting  agent 
of  influenza,  and  developed  it  in  pure  culture.  The  state- 
ments of  Pfeiffer  have  since  been  completely  confirmed. 

Morphology  of  the  Influenza-bacillus. — The  influenza- 
bacillus  is  an  extremely  small  organism  (0.2  //  thick  and 
0.5  ;a  long),  in  thickness  not  quite  equaling  the  slender  bacilli 
of  mouse-septicemia,  and  being  only  twice  or  thrice  as  long  as 
wide.  Its  extremities  are  rounded.  Rarely  in  the  sputum  and 
more  commonly  in  recent  pure  culture,  the  bacilli  form  short 
pseudo-filaments.  Long  bands  in  cultures  three  or  four  days 
old  are  to  be  considered  as  beginning  involution-manifestations. 
The  influenza-bacilli  possess  no  capsule,  and  are  without  move- 
ment of  their  own.  Frequently,  two  especially  short  bacilli  lie 
close  to  each  other  (division-forms).  This  may  readily  give 
rise  to  confusion  with  diplococci. 

Influenza-bacilli  appear  not  to  possess  spores.  Spore-like 
formations  have  never  been  found  in  the  secretions  or  in  cul- 
tures, and,  besides,  the  bacillus  is  but  little  resistant  to  the  influ- 
ence of  temperature,  drying,  etc. 

Staining  of  Influenza-bacilli. — The  bacilli  take  the  stain 
with  considerable  difficulty.  Loffler's  solution  of  methylene- 
blue  may  be  employed,  and,  still  better,  a  dilute,  pale-red  solu- 
tion of  carbolfuchsin  in  water.  The  preparation  must  be  ex- 
posed to  the  action  of  the  stain  for  from  five  to  ten  minutes. 
If  the  exposure  is  of  shorter  duration,  or  if  other  stains  are 
employed,  the  central  portion  of  the  bacillus  is  often  more 
feebly  stained  than  the  extremities.  The  bacilli  are  not  stained 
by  Gram's  method. 

Cultivation  of  the  Influenza-bacillus. — The  influenza- 
bacillus  is  strictly  aerobic,  and  it  develops  only  in  the  presence 
of  hemoglobin  or  of  leukocytes.  The  latter  fact  explains  why 
cultivation  of  the  influenza-bacilli  remained  for  so  long  a  time 


282  CLINICAL  BACTERIOLOGY. 

unsuccessful.  Pfeiffer  also  was  often  able  to  cultivate  the 
bacilli  from  the  sputum  or  pus  from  the  lungs  directly  upon 
agar,  but  this  did  not  take  place  invariably,  and  at  times  it  was 
quite  impossible  to  continue  the  growth  of  such  cultures  in  any 
way.  The  explanation  for  this  fact  is  that  the  bacilli  developed 
in  the  first  culture  when  with  the  infecting  material  a  trace  of 
blood  was  simultaneously  transferred  to  the  culture-medium. 
Growth  failed  to  take  place,  however,  when  the  blood  was 
wanting,  and  thus  also  in  all  daughter-cultures. 

The  development  of  influenza-bacilli  takes  place  regularly, 
and  the  culture  obtained  may  be  continued  indefinitely,  if  the 
infecting  material  is  inoculated  upon  a  culture-medium  contain- 
ing blood,  and  best  upon  blood-agar  tubes  (p.  82).     For  the 


\ 


Fig.  62. — Bacillus  influenzae,  from  a  gelatin-culture;  X  looo  (Itzerott  and  Niemann). 

development  of  pure  cultures  Pfeiffer  recommends  the  following 
method  :  The  infecting  material — bronchial  sputum  or  fluid  from 
a  bronchopneumonic  infiltrated  portion  of  the  lung  in  a  case 
of  influenza-pneumonia — is  rubbed  up  with  one  or  two  cubic 
centimeters  of  bouillon  to  a  homogeneous  emulsion.  By  means 
of  a  platinum  loop  some  of  this  is  inoculated  upon  blood-agar, 
and  also  for  control-purposes  upon  ordinary  glycerin-agar,  tubes, 
care  being  taken  to  secure  uniform  distribution  of  the  infecting 
material  upon  the  entire  surface.  The  dilution  of  the  sputum 
in  the  bouillon  is  intended,  in  the  first  place,  to  separate  the 
influenza-bacilli  to  such  a  degree  that  isolated  colonies  will  form 
upon  the  blood-agar  tubes.  In  the  second  place  the  hemoglo- 
bin that  may  be  present  in  the  infecting  material  will  be  so 
greatly  diluted  that  the  influenza-bacilli  will  be  incapable  of 


INFLUENZA.  283 

developing  in  the  control-tubes  not  previously  treated  with 
blood.  The  inoculated  test-tubes  are  placed  in  the  thermostat. 
After  the  lapse  of  twenty-four  hours  the  influenza-colonies  will 
become  visible  in  the  blood-agar  tubes  as  densely  packed, 
watery  drops,  whereas  the  control-tubes  will  be  either  sterile 
or  present  isolated  colonies  of  streptococci,  diplococci,  or  other 
bacteria,  which  were  present  in  the  infecting  material  in  addi- 
tion to  the  influenza- bacilli. 

The  water-like  drops  of  influenza-colonies  are  usually  so  small 
that  they  are  only  visible  with  the  aid  of  a  lens.  They  exhibit 
little  tendency  to  become  confluent.  Should  they  be  especially 
numerous  and  close  together,  they  coalesce  to  form  larger  drops 
with  curved  margins,  but  these  permit  a  recognition  of  the  aggre- 
gation of  individual  colonies.  If  the  colonies  are  isolated  and 
widely  separated  from  one  another,  they  may  grow  to  the  size 
of  a  pinhead,  but  in  this  case  also  they  retain  a  vitreous  trans- 
parency. The  water  of  condensation  of  influenza-cultures  gen- 
erally remains  clear.  When  mixed  with  blood  derived  from  the 
oblique  surface  of  the  culture,  delicate  white  flocculi  form  in  it. 

In  bouillon  mixed  with  blood  and  spread  in  a  thin  layer  the 
influenza-bacillus  thrives  quite  abundantly. 

The  plate-procedure  is  greatly  to  be  recommended  for  the  iso- 
lation of  influenza-bacilli  and  for  diagnostic  purposes,  if  some 
blood  is  added  to  the  liquefied  agar  before  inoculation,  or 
if  agar  is  permitted  to  solidify  in  Petri  dishes  and  blood  is 
added,  and  the  diluted  sputum,  etc.,  is  smeared  upon  the  sur- 
face in  several  streaks.  The  colonies  present  the  same  appear- 
ance as  those  in  agar-tubes. 

The  temp&rature-opti7num  for  the  cultivation  of  influenza- 
bacilli  is  that  of  the  body.  The  upper  limit  for  growth  is  42° 
C.  (107.6°  F.),  the  lower  between  26°  C.  (78.8°  F.)  and  27° 
C.   (80.6°  F.).     The  bacilli  do  not  grow  at  room-temperature. 

Oxygen  is  always  necessary  for  the  development  of  the  influ- 
enza-bacilli ;  they  do  not  grow  in  an  atmosphere  of  hydrogen 
or  of  carbon  monoxid,  even  in  the  presence  of  blood. 

Pfeiffer  undertook  to  determine  what  constituent  of  the  blood 
the  influenza-bacilli  require  for  their  development.  On  making 
transfers  from  blood-serum  or  blood-fibrin  to  agar-tubes  no 
growth  took  place.  Red  blood-corpuscles  were  invariably 
necessary,  and  particularly  the  hemoglobin  contained  in  them, 
as  was  later  shown,  was  the  active  substance.  Hemoglobin- 
agar  (p.  82)  is  equally  adapted  with  blood-agar  for  the  cultiva- 
tion of  influenza-bacilli.  Pfeiffer  endeavored  to  associate  this 
Indispensability  of  hemoglobin  for  the  growth  of  influenza- 
bacilli  at  first  with  its  relations  to  oxygen,  with  its  faculty  of 
acting  as  an  oxygen -carrier.  He  succeeded,  however,  in  ob- 
taining growth  upon  an  agar-layer  in  the  presence  of  carbon- 


284  CLINICAL   BACTERIOLOGY. 

monoxid  hemoglobin.  Exposure  of  blood-agar  tubes  to  a  tem- 
perature of  70°  C.  (158°  F. ),  and  even  boiling  of  the  hemo- 
globin, failed  to  prevent  entirely  the  development  of  the  in- 
fluenza-bacilli. Pfeiffer  was  then  led  to  believe  that  the  iron- 
content  of  the  hemoglobin  was  the  important  factor,  but  he  was 
unable  to  cultivate  the  bacilli  in  culture-media  containing  iron 
other  than  that  of  the  blood. 

It  may  be  mentioned  further  that  all  kinds  of  blood  exhibit 
the  same  specific  activity  with  relation  to  influenza-bacilli. 
Pfeifl'er  obtained  growth  upon  the  blood  of  rabbits,  guinea- 
pigs,  pigeons,  and  fish,  and  in  more  luxuriant  degree  and  more 
speedily  on  pigeon's  blood — which  is  rich  in  hemoglobin — than 
on  human  blood. 

Resistance  of  Influenza-bacilli. — Influenza-bacilli  are 
destroyed  in  a  kw  minutes  when  exposed  to  a  temperature 
of  60°  C.  (140°  F.).  They  cease  to  develop  at  a  temper- 
ature of  43°  C.  (109.4°  F.),  but  they  are  only  coagulated, 
not  destroyed,  for  if  tubes  that  have  been  exposed  for 
forty-eight  hours  to  a  temperature  of  43°  C.  (109.4°  F.), 
and  have  remained  sterile,  are  then  exposed  to  a  tempera- 
ture of  37°  C.  (98.6°  F.),  colonies  will  yet  develop  abun- 
dantly. In  unsterilized  drinking-water  the  bacilli  die 
quickly — in  from  twenty-four  to  thirty-six  hours.  Upon 
blood-agar  and  in  bouillon  they  retain  their  vitality  for  from 
fourteen  to  eighteen  days,  and  in  moist  sputum  they  appear 
to  preserve  their  infectivity  for  at  least  fourteen  days.  The 
influenza-bacilli  are  quite  sensitive  to  drying.  When  dried 
in  blood  or  sputum  at  a  temperature  of  37°  C.  (98.6°  F.), 
they  succumb  in  an  hour  or  two,  and  when  dried  at  room- 
temperature,  within  not  more  than  from  thirty-six  to  forty 
hours. 

Occurrence  of  Influenza-bacilli. — The  influenza-bacilli 
occur  regularly  in  the  secretions  of  influenza-patients.  In 
the  secretion  of  the  nasal  cavities  the  specific  bacilli  have 
been  found  in  enormous  numbers ;  although  generally 
associated  with  other  microorganisms,  yet,  however,  in 
preponderating  number.  The  secretion  in  a  case  of  ordi- 
nary coryza,  on  the  other  hand,  is  remarkably  free  of  bac- 
teria, being  almost  sterile.  The  sputum  in  cases  of  bron- 
chitis and  pneumonia  complicating  influenza  is  viscid, 
mucopurulent,  globular,  and  not  seldom  also  purulent  and 
confluent,  in  color  often  yellowish  green,  not  rarely  pure 
white  and  only  seldom  rusty  brown,  and  it  contains  the 


INFLUENZA.  .  285 

influenza-bacilli  in  almost  absolutely  pure  culture,  and 
always  in  surprising  number.  The  bacilli  usually  lie  in  the 
mucous  ground-substance  arranged  in  nests  and  groups  ; 
they  are  found  also  within  the  pus-corpuscles,  at  the  begin- 
ning of  the  disease  in  small  number,  and  during  convales- 
cence in  preponderating  number.  In  the  latter  condition 
they  surround  the  nucleus  and  are  not  included  within 
it.  Sputa  containing  influenza-bacilli  are  often  ejected  for 
days  and  months.  Especially  in  cases  of  tuberculosis  have 
such  chronic  complications  of  influenza  with  broncho- 
pneumonic  localization  not  been  rare.  The  bacilli  have 
been  found  in  the  bronchopneumonic  foci  in  cases  of 
influenza-pneumonia,  rarely  in  the  pus  in  cases  of  influenza- 
empyema.  Canon  obsei-ved  in  the  blood  of  influenza- 
patients  delicate  bacilli  resembling  those  of  influenza. 
According  to  Pfeiffer's  investigations,  these  organisms,  if 
influenza-bacilli  at  all,  are  exceptional,  for,  as  a  rule,  the 
organisms  are  not  present  in  the  blood. 

With  regard  to  the  localization  of  the  bacilli  in  the  gastric 
and  nervous  forms  of  influenza,  unequivocal  investigations 
are  wanting.  Likewise,  the  numerous  complications  and 
sequelae  of  influenza  have  thus  far  been  little  studied  from 
the  bacteriologic  standpoint,  so  that  it  has  not  yet  been  de- 
termined whether  they  represent  results  of  the  activity  of 
the  influenza-bacillus  or  its  toxin,  or  are  secondary  infec- 
tions. In  a  case  of  influenza-aortitis  Pfeiffer  found,  in  addi- 
tion to  the  diplococci  of  Frankel,  numerous  influenza- 
bacilli,  and  in  the  exudate  from  a  case  of  influenza-meningitis 
diplococci  exclusively. 

The  bacillus  of  influenza  has  never  been  found  outside 
the  human  body,  in  the  earth,  or  in  water.  It  could  scarcely 
persist  for  a  long  time  under  these  conditions  on  account  of 
its  feeble  powers  of  resistance. 

Portals  of  Infection  for,  and  Distribution  of,  the  In- 
fluenza-bacillus.— The  influenza-bacillus  is  probably  taken 
up  by  the  air-passages  exclusively.  Its  distribution  by 
means  of  dried  and  powdered  sputum  can  play  an  etiologic 
role  only  in  restricted  degree,  as  the  bacillus  withstands 
drying  so  badly.  The  ordinary  mode  of  conveyance  is 
certainly  by  means  of  the  moist  nasal  and  bronchial  secre- 
tions of  influenza-patients.  The  widespread  and  often  pan- 
demic distribution  of  influenza  may  be  explained  by  the  fact 
that  in  the  first  place  the  susceptibility  of  human  beings  to 


286  .    CLINICAL  BACTERIOLOGY. 

the  disease  is  quite  considerable ;  further,  that  the  disease 
appears  in  many  cases  in  the  form  of  a  mild  coryza  and  of  a 
harmless  bronchial  catarrh,  and  these  at  the  beginning  of  the 
epidemic  are  not  immediately  recognized  as  influenza  ;  that 
even  after  the  epidemic  character  of  the  disease  is  established 
they  do  not  confine  patients  to  the  house,  so  that  opportunity 
is  thus  afforded,  in  sneezing  and  in  coughing,  to  disseminate 
innumerable  influenza-bacilli  among  those  not  yet  infected. 
The  sudden  recrudescence  of  apparently  extinct  epidemics 
is  rendered  comprehensible  by  the  fact  just  mentioned,  that 
there  are  cases  of  chronic  influenza  that  act  for  a  long  time 
as  carriers  of  bacilli  capable  of  inducing  influenza. 

Experiments  on  Animals. — Even  in  the  most  exten- 
sive epidemics  of  influenza  domestic  animals  escape  the  dis- 
ease. Success  in  the  transmission  of  influenza-bacilli  to 
animals  was  therefore  improbable  from  the  outset.  Pfeififer 
undertook  experiments  on  mice,  rats,  guinea-pigs,  rabbits, 
swine,  cats,  dogs,  and  monkeys.  Only  in  the  last-named 
animal  was  it  possible  to  induce  an  infection  resembling  in- 
fluenza, and  then  only  by  introducing  the  bacteria  through 
the  chest-wall  directly  into  the  lung,  and  also — which  was 
more  important^in  a  monkey,  by  introducing  an  influenza- 
culture  into  the  uninjured  nose.  The  disease  manifested 
itself  by  some  cough  and  fever  of  several  days'  duration. 
Multiplication  of  the  inoculated  bacteria  was,  however,  not 
observed.  By  introducing  large  amounts  death  can  be 
caused  in  rabbits  quite  rapidly,  with  antemortem  depres- 
sion of  temperature  (to  32.2°  C. — 90°  F.) ;  under  these 
circumstances  the  symptoms  are  probably  the  result  of  in- 
toxication. Symptoms  of  intoxication  (fever,  muscular 
paresis)  also  appeared  in  rabbits  after  intravenous  injection 
of  considerable  amounts  of  bacteria.  Further,  cultures  de- 
vitalized by  chloroform  proved  quite  toxic.  The  influenza- 
bacillus  thus  appears  to  generate  an  active  toxin,  a  fact  that 
sheds  considerable  light  upon  the  nervous  manifestations 
frequently  observed  in  cases  of  influenza  in  human  beings. 

Immunity. — The  monkeys  in  Pfeiffer's  experiments  re- 
acted much  less  vigorously  to  a  second  injection  of  influenza- 
bacilli  than  to  the  first ;  and  Pfeiffer  believes  this  to  be  an 
indication  of  immunity.  Human  beings  certainly  can  be 
attacked  several  times  by  influenza,  even  in  the  course  of 
the  same  epidemic.  This  fact  does  not,  of  course,  exclude 
the  possibility  that  a  certain  degree  of  immunity  follows  an 


ANTHRAX.  287 

attack  of  influenza,  but  this  must,  however,  be  considered 
as  only  temporary. 

Pseudo-influenza-bacilli. — In  a  number  of  broncho- 
pneumonic  foci  in  patients  not  suffering  from  influenza  (but 
from  diphtheria)  Pfeififer  found  bacilH  that  in  form  and  stain- 
ing properties  resembled  influenza-bacilli,  and  that,  like 
these,  developed  exclusively  upon  blood-agar.  Similar 
bacilli  have  since  been  isolated  by  various  observers  from 
cases  of  otitis  media  and  of  influenza.  Pfeiffer  believes  these 
organisms  to  be  related  to  influenza-bacilli,  and  he  desig- 
nates them  pseudo-influenza-bacilli.  They  are  to  be  dis- 
tinguished from  true  influenza-bacilli  by  culture,  in  which, 
after  the  lapse  of  twenty-four  hours,  they  appear  consider- 
ably larger  in  all  dimensions,  and  they  exhibit  a  marked 
tendency  to  the  formation  of  long  pseudo-filaments.  In 
similar  cultures  of  the  true  bacillus  the  latter  are  wanting 
entirely  or  appear  but  exceptionally. 

Bacteriologic  Diagnosis  of  Influenza. — The  bacterio^ 
logic  diagnosis  is  made  from  examination  of  the  sputum 
derived  from  the  deepest  possible  portions  of  the  air- 
passages,  and  thus  preferably  from  examination  of  the 
bronchial  sputum.  Microscopic  examination  alone  is  usu- 
ally insufficient,  but,  as  a  rule,  cultivation  of  the  bacillus  is 
necessary,  the  method  for  which  has  already  been  described 
in  detail  (p.  283,  Plate-procedure).  Bacteriologic  examina- 
tion may  acquire  differential  diagnostic  significance.  Thus, 
Borchardt  relates  a  case  in  which  the  diagnosis  oscillated 
for  a  long  time  between  typhoid  fever  and  influenza,  until 
bacteriologic  examination  decided  in  favor  of  the  latter. 


ANTHRAX. 

Pollender,  in  Germany,  in  1849,  and  Rayer  and  Davaine, 
in  France,  in  1850,  were  the  first,  independently  of  each 
other,  to  detect  bacilli  in  the  blood  of  animals  suffering 
from  anthrax.  The  growth  of  the  anthrax-bacillus  in 
pure  culture  and  the  experimental  development  of  the  dis- 
ease by  means  of  the  bacillus  were  successfully  accom- 
plished first  by  Koch  in  1876. 

Morphology  of  the  Anthrax-bacilli. — Anthrax-bacilli 
appear  as  transparent,  homogeneous,  nonmotile  rods.  They 
are  from  i  to  i-S  fJ-  thick,  and  from  5  or  6  to  10 /x  long,  but  they 


288  CLINICAL  BACTERIOLOGY. 

are  subject  to  great  variations  in  size.  In  cultures  the  bacillus 
is  prone  to  be  considerably  longer  than  in  the  animal  organism. 
In  the  blood  of  human  beings  it  is  shorter  than  in  that  of  ro- 
dents; in  cattle  it  is  shorter  than  in  white  mice  and  in  guinea- 
pigs.  The  broad  side  of  the  bacillus  is  slightly  rounded.  The 
surfaces  of  two  adjacent  bacilli  in  direct  contact  are,  however, 
plane.  In  the  blood  of  animals  suffering  from  anthrax  the 
bacilli  are  at  times  collected  into  small  filaments  of  two  or 
four,  or  at  most  five,  members.  Only  attenuated  bacilli,  just 
capable  of  causing  death  in  experimental  animals,  form  long 
filaments  in  the  organs  of  the  animals.  Such  filaments  are 
usual  in  the  body  of  the  frog.     In  cultures,  on  the  other  hand, 


Fig.  63. — Bacillus  antliracis,  stained  to  show  the  spores ;  X  1000 
(Frankel  and  PfeiflFer). 

the  anthrax-bacillus  exhibits  an  especial  tendency  to  the  forma- 
tion of  long,  intertwining  chains.  Frequently,  bacilli  obtained 
from  the  lesions  of  the  disease  present  a  bright  border  that 
some  observers  have  looked  upon  as  a  capsule.  This  supposed 
capsule  may  be  demonstrated  by  the  staining  method  of  Johne 
in  the  following  manner:  The  preparation  made  from  the  blood 
or  visceral  fluid  and  dried  in  the  air  is  passed  three  times 
through  the  flame ;  stained  for  from  fifteen  to  thirty  seconds 
in  a  slightly  warmed,  two  per  cent,  aqueous  solution  of 
gentian-violet ;  washed  in  water ;  exposed  for  ten  minutes  to 
the  action  of  a  one  per  cent,  solution  of  acetic  acid  ;  and  ex- 
amined in  water.  It  is  important  to  make  the  examination  in 
water,  as  the  mucoid  capsule  is  not  visible  when  the  specimen  is 


ANTHRAX.  289 

mounted  in  Canada  balsam.  The  bacillus  can  be  stained  with 
all  aniline  dyes  and  also  by  the  method  of  Gram. 

Cultivation  of  Anthrax-bacilli. — The  anthrax-bacillus  is 
extremely  indifferent  with  regard  to  its  nutrient  material.  It 
will  develop  in  the  absence  of  oxygen,  but  it  does  not  then  gen- 
erate a  peptonizing  ferment.  The  temperature-minimum  is  12° 
C.  (53.6°  F.);  the  temperature-optimum,  35°  C.  (95°  F.)  ; 
the  temperature-maximum,  45°  C.  (113°  F. ). 

On  gelatin-plates,  with  a  magnification  of  from  80  to  100, 
the  superficial  colonies  appear  as  round  discs  of  yellowish  color, 
constituted  of  a  tangle  of  threads  which  form  a  dense,  impene- 
trable convolution  at  the  center.    Especially  the  border  presents 


Fig.  64. — Bacillus  anthracis:  colony  three  days  old  upon  a  gelatin-plate;  impress- 
preparation;  X  1000  (Frankeland  Pfeiflfer). 

quite  distinctly  this  filamentous  structure,  and  from  it  fre- 
quently passes  off  a  delicate  network  of  convoluted  and  curled 
processes,  which  gives  the  colonies  their  characteristic  appear- 
ance. The  gelatin  in  the  neighborhood  is  softened,  and  begins 
slowly  to  undergo  liquefaction. 

In  gelatin  stab-cultures  the  line  of  inoculation  forms  a  whitish 
band,  from  the  circumference  of  which  numerous  ramifying 
processes  extend  into  the  gelatin.  Liquefaction  of  the  culture- 
medium  progresses  slowly,  the  nonmotile  bacilli,  by  reason  of 
their  weight,  sinking  to  the  bottom  of  the  liquefied  area. 

In  bouillon  small  fragments  appear  and  fall  to   the  bottom  ; 
they  represent  a  union  of  the  anthrax-bacilli  for  the  formation 
of  convolutions  of  filaments. 
19 


290  CLINICAL  BACTERIOLOGY. 

On  agar-agar  a  dense,  creamy,  coherent  deposit  forms ;  and 
Vi^on  potatoes  a  dry,  whitish-gray  layer.  Blood-serum  is  lique- 
fied.    Mi  Ik  is  coagulated  and  peptonized. 

Sporulation. — If  anthrax-cultures  are  exposed  to  tempera- 
tures above  i8°  C.  (64.4°  F.),  the  bacilli  soon  exhibit  spore- 
formation.  Slight  development  of  spores  takes  place,  besides, 
down  to  the  temperature-minimum.  The  anthrax-spore  in- 
variably lies  exactly  in  the  middle  of  the  mother-cell,  being 
much  shorter,  but  as  wide,  and  possessing  an  oval  shape. 
After  a  time  the  germinating  bacillus  disintegrates,  and  the 
spore  is  set  free.  If  the  spore  is  introduced  into  a  sterile 
hanging  drop  of  bouillon,  gelatin,  or  agar,  its  germination  can 
be  accurately  followed  under  the  microscope  (possibly  with  the 
employment  of  a  warm  stage).  The  spore  first  loses  its  glis- 
tening appearance,  and  increases  in  volume.  Its  membrane 
then  ruptures  at  one  extremity  and  permits  the  escape  of  the 
newly  formed  bacillus.  The  young  bacillus  enlarges  in  the 
direction  of  the  long  axis  of  the  spore,  and  soon  throws  off  the 
still  adherent  spore-membrane.  Spore-formation  takes  place 
only  in  the  presence  of  free  oxygen — thus  never  in  the*animal 
body  and  never  in  the  uninjured  cadaver. 

The  higher  the  temperature  the  more  rapidly  do  the  spores  de- 
velop— at  37°  C.  (98.6°  F.),  as  early  as  twenty  hours;  at  21° 
C.  (69.8°  F.),  not  before  seventy-two  hours;  while  above  42° 
C.  (107.6°  F.)  the  anthrax-bacilli  do  not  generate  spores  at 
all.  It  is  possible  artificially  to  deprive  the  anthrax-bacilli  of 
their  capability  of  generating  spores.  For  this  purpose  it  is 
only  necessary  to  add  certain  antiseptic  substances  to  the  nutri- 
ent medium  (Chamberland  and  Roux)  :  for  instance,  carbolic 
acid  in  the  proportion  of  from  i  :  600  to  i  :  1000,  potassium 
bichromate  in  the  proportion  of  i  :  2000.  The  bacilli  culti- 
vated upon  such  nutrient  media  remain  sporeless  permanently  in 
all  subsequent  generations,  and  in  this  way  special  asporogenous 
anthrax-bacilli  are  developed. 

Resistance  of  Anthrax-bacilli  and  of  Anthrax- spores. 

— Anthrax-spores,  like  all  other  spores,  are  exceedingly  re- 
sistant structures  ;  while  the  fully  developed  bacilli  suc- 
cumb after  exposure  for  a  quarter  of  an  hour  to  tempera- 
tures in  the  neighborhood  of  60°  C.  (140°  F.),  the  spores 
die  in  compressed  steam  at  a  temperature  of  107°  C.  (224.6° 
F.)  only  after  the  lapse  of  five  minutes  ;  and  in  live  steam 
at  a  temperature  of  100°  C.  (212°  F.)  only  after  from  twelve 
to  fifteen  minutes.  Five  per  cent,  carbolic  acid  destroys  the 
mature  forms  of  anthrax-bacilli  in  ten  seconds,  but  the 
spores   not  before  from    thirty-seven    to    forty    days.     In 


ANTHRAX.  291 

I  :  looo  solutions  of  mercuric  chlorid  the  spores  are  not 
destroyed  earlier  than  after  the  lapse  of  twenty  hours  (Gep- 
pert).  In  sterilized  distilled  water  or  tap-water  anthrax- 
bacilli  survive  only  for  three  days,  but  the  spores  for  from 
one  hundred  and  fifty-four  days  to  a  year.  The  lower,  fur- 
ther, the  temperature,  the  better  do  the  anthrax-organisms 
withstand  the  injurious  influence  of  the  water.  Anthrax- 
bacilli  in  distilled  and  other  water  have  been  demonstrated 
to  undergo  sporulation  at  a  temperature  of  20°  C.  (6S°  F.). 
The  bacilli  are  rapidly  destroyed  by  the  influence  of  putre- 
faction, while  the  spores  have  been  found  alive  in  putrefac- 
tive mixtures  after  a  month. 

The  statements  made  apply  only  to  robust  spores,  the 
descendants  of  highly  virulent  bacilli.  Anthrax-spores 
of  indifferent  source  do  not  invariably  react  alike ;  thus, 
von  Esmarch  was  able  to  render  some  anthrax-bacilli  in- 
nocuous by  means  of  5  per  cent,  carbolic  acid  within  two 
days,  and  by  means  of  live  steam  at  a  temperature  of  100° 
C.  (212°  F.)  within  three  minutes. 

The  Occurrence  of  Anthrax  in  Animals. — The  animal 
most  susceptible  to  anthrax  is  the  sheep,  and  it  is  often 
attacked  by  the  disease  spontaneously.  There  is,  however, 
one  variety  of  sheep  that  is  immune — namely,  the  Algerian 
sheep.  This  immunity  is  not  dependent  upon  climatic  con- 
ditions, for  European  sheep  transported  to  Algiers  were  as 
readily  attacked  by  anthrax  as  at  home.  White  mice  and 
guinea-pigs  succumb  likewise  with  regularity  to  experi- 
mental anthrax-infection  ;  rabbits  are  somewhat  more  resist- 
ant ;  but  none  of  these  three  species  of  animals  is  scarcely 
ever  exposed  to  natural  infection.  Cows  and  calves  not 
rarely  succumb  to  spontaneous  anthrax,  although  they 
prove  rather  refractory  to  experimental  subcutaneous  infec- 
tion. Horses,  antelope,  deer,  goats,  at  times  acquire  an- 
thrax ;  swine  more  rarely.  With  regard  to  the  much- 
discussed  immunity  of  white  rats  to  anthrax,  it  appears 
that  young  animals  die  of  anthrax  after  experimental 
infection,  whereas  old  animals  usually  exhibit  only  a 
local  lesion,  from  which  they  recover.  Carnivorous  ani- 
mals (dogs,  cats,  etc.)  rarely  are  attacked  spontaneously 
by  anthrax.  Experimentally,  the  disease  can  be  induced 
in  mature  animals  only  by  intravenous  injection  of  the 
bacilli.  New-born  or  quite  young  dogs,  on  the  other  hand, 
prove  extremely  susceptible.     Birds,  reptiles,  batrachians, 


292  CLINICAL   BACTERIOLOGY. 

possess  a  considerable  degree  of  immunity  to  anthrax.  If, 
however,  on  the  one  hand,  the  high  temperature  of  birds  is 
reduced  artificially ;  or  if,  upon  the  other  hand,  the  low 
temperature  of  reptiles  is  elevated  artificially,  these  animals 
react  precisely  like  other  animals — that  is,  they  succumb  to 
inoculation  with  anthrax.  Pigeons  may  also  be  rendered 
susceptible  to  anthrax-infection  by  starvation. 

Occurrence  of  Anthrax  in  Human  Beings. — Human 
beings  are  infected,  as  a  rule,  through  contact  with  animals 
suffering  from  anthrax,  and  with  the  cadavers  of  those  dead 
of  the  disease.  Not  only  the  fresh  cadaver  is  infectious, 
but  also,  owing  to  the  presence  of  spores,  which  develop 
rapidly  in  summer  with  unobstructed  access  of  oxygen, 
every  individual  portion  of  the  body — wool,  hair,  horns, 
etc. — -even  after  the  lapse  of  a  long  time.  For  this  reason 
tanners  are  exposed  to  danger  in  manipulating  the  skins  of 
animals  dead  of  anthrax.  Even  after  the  hide  has  been 
tanned,  the  spores  are  not  always  destroyed  with  certainty, 
and  shoemakers,  furriers,  and  harness-makers  have  been 
known  to  be  infected  by  means  of  such  leather.  Workers 
in  horsehair,  turners  of  horn,  brush-makers,  dealers  in 
hides,  etc.,  are  likewise  exposed  to  the  danger  of  infection 
with  anthrax  in  so  far  as  the  raw  material  with  which  they 
work  is  derived  from  animals  suffering  from  anthrax.  The 
following  instructive  example,  in  this  connection,  has  been 
reported  by  Einike  :  An  ox  dies  of  anthrax.  Two  indi- 
viduals who  partake  of  its  meat  die  of  the  same  disease. 
The  hide  of  the  animal,  after  it  has  been  macerated  for  a 
time  in  a  small  lake,  is  used  by  a  harness-maker  for  making 
halters.  This  man  is  attacked  by  anthrax.  The  two 
horses  that  wear  the  halters  likewise  succumb  to  this 
disease.  Of  a  herd  of  sheep  that  bathe  in  the  small  lake 
mentioned  twenty  are  attacked  by  anthrax. 

The  transmission  of  anthrax  from  animals  to  human 
beings  may  further  be  effected  by  certain  varieties  of  flies 
that  possess  a  stiff  and  pointed  sting  (stomoxys,  glos- 
sina,  Ixodes).  In  the  bodies  of  such  insects  as  have  fed 
upon  anthrax-cadavers  anthrax-bacilli  have  been  repeatedly 
demonstrated. 

Human  beings  are  not  especially  susceptible  to  anthrax. 
For  this  reason  only  a  local  lesion  generally  forms  at  first — 
the  so-called  mali£^?iant  pus tti/c  (a.nthra.x-ca.rhunc\e) — which 
frequently  terminates  in  recovery.     In  other  cases  the  car- 


ANTHRAX.  293 

buncle  is  succeeded  by  septicemic  general  infection,  leading 
to  death.  Anthrax  may  be  further  transmitted  from  one 
human  being  to  another.  In  the  nature  of  things  such  an 
occurrence  is  rare,  although  it  has  been  reliably  observed. 
Jacobi  has  reported  four  cases  in  which  the  disease  was 
conveyed  by  means  of  a  hypodermic  syringe  that  had  pre- 
viously been  employed  in  a  case  of  anthrax. 

The  infectivity  of  malignant  pustule  was  formerly  denied, 
and  upon  the  basis  of  experiments  in  which  the  serous  con- 
tents of  the  pustules  were  inoculated  in  healthy  individuals, 
without  the  development  of  any  reaction  whatever.  Such 
experiments  are,  however,  not  conclusive,  as  the  serous 
contents  of  the  vesicle,  which  surround  the  central  eschar, 
contain  only  a  small  number  of  bacilli. 

Natural  Portals  of  Entry  for  the  Anthrax-bacillus. — 

(a)  Skin. — Infection  through  the  skin  is  possible  only  in 
the  presence  of  a  breach  in  continuity,  however  slight.  In 
human  beings  this  mode  of  infection  is  the  most  common, 
and  particularly  in  individuals  that  come  in  contact  with 
anthrax-cadavers  or  constituent  parts  thereof  The  course 
of  infection  has  already  been  outlined.  At  the  point  of 
infection  a  carbuncle  develops,  and  this  leads  either  to 
recovery  or  to  general  infection. 

(U)  Digestive  Tract. — Spontaneous  anthrax  of  herbivor- 
ous animals  almost  always  involves  the  intestine  (pasture- 
anthrax).  If  the  herds  graze  on  some  meadow-lands  in 
certain  districts  (champs  maudits),  anthrax  is  sure  to 
occur  among  them.  In  Germany  such  regions  exist  in 
some  parts  of  Saxony  and  in  the  Bavarian  Alps,  in  France 
especially  in  the  district  Beauce,  in  Austria  in  the  Hun- 
garian lowlands,  and  in  Russia  in  the  neighborhood  of 
Novgorod.  It  is  believed  that  the  earth  in  these  meadow- 
lands  contains  spores,  which  are  swallowed  by  the  animals 
in  grazing.  The  question  naturally  arises.  How  are  the 
anthrax-spores  brought  to  the  surface  of  these  fields  ? 
Pasteur,  who  was  the  first  to  occupy  himself  with  this 
question,  found  anthrax-spores  in  the  earth  of  graves  in 
which  years  before  anthrax-cadavers  had  been. buried.  It 
was  his  opinion  that  these  spores  are  carried  to  the  surface 
from  the  depth  by  earth-worms.  The  worms  swallow  the 
contaminated  earth,  later  crawl  upward  to  the  surface,  and 
there  deposit  the  spores  with  their  excrement.  Such  a 
possibility  has  been  demonstrated  experimentally,  and  that 


294  CLINICAL  BACTERIOLOGY. 

it  may  actually  be  realized  would  appear  from  the  observa- 
tions of  Bollinger,  who  succeeded  in  finding  anthrax-spores 
in  the  bodies  of  worms  collected  from  the  anthrax-fields  of 
the  Bavarian  Alps.  The  view  of  Pasteur  was  vigorously 
attacked  by  Koch.  At  the  depth  at  which  anthrax-cadavers 
are  usually  buried  (from  i/^  to  i  meter)  the  temperature, 
even  in  the  hottest  summer-months,  is  only  between  14°  C. 
(57.2°  F.)  and  18°  C.  (64.4°  R),  a  temperature  that  is 
extremely  unfavorable  for  sporulation.  According  to 
Soyka,  the  addition  of  porous  particles  of  earth  to  artificial 
cultures  furthers  spore -production  quite  materially.  Then, 
it  is  further  to  be  borne  in  mind  that  the  temperature  of  the 
buried  cadavers  is  certainly  raised  somewhat  in  consequence 
of  the  putrefactive  processes  taking  place.  Kitasato  buried 
gelatin-cultures  and  agar-cultures  of  anthrax  in  the  earth 
and  showed  that  at  a  depth  of  one  meter  sporulation  pro- 
ceeded in  the  months  of  June,  July,  and  August.  The 
conditions  provided  experimentally  and  artificially  are,  how- 
ever, not  entirely  comparable  with  those  observed  prac- 
tically. Animals  dead  of  anthrax  are,  or  were  formerly, 
first  dissected  and  skinned,  the  secretions,  the  blood,  etc., 
being  spilled  indiscriminately ;  burial  also  is  not  effected 
immediately ;  in  brief,  abundant  time  and  opportunity  are 
afforded  the  bacilli,  at  least  in  summer,  to  form  spores. 
The  cadaver,  which  is  now  buried,  contains  not  only 
mature  forms,  but  also  permanent  forms ;  quite  apart 
from  the  fact  that  numerous  anthrax-bacilli  are  already 
present  upon  the  surface  of  the  earth  in  consequence  of  the 
various  manipulations  at  the  place  where  the  autopsy  was 
held.  It  is  true  that  if  the  animal  is  buried  at  a  depth  of 
two  or  three  meters  immediately  after  death,  without  further 
manipulation,  the  formation  of  spores  would  certainly  be 
entirely  prevented ;  for,  then,  one  of  the  most  important 
conditions  for  spore-formation — namely,  unobstructed  ac- 
cess of  oxygen — would  be  completely  wanting. 

In  addition  to  earth-worms  anthrax-spores  may  be  dis- 
seminated by  means  of  snails,  flies,  etc.  Further,  the 
diseased  animal  furnishes  during  life  sufficient  material  for 
subsequent  infection  through  the  feces  and  the  urine,  which 
contain  the  bacilli  in  large  number.  In  these,  and  also  in 
vegetable  culture -media,  the  bacilli,  which  are  so  indifferent 
with  relation  to  the  nature  of  the  nutrient  material,  multiply 
abundantly,  and  in  summer  also  form  spores.     The  excre- 


ANTHRAX.  295 

ment  of  animals  fed  with  anthrax-matter  contains  spores 
regularly.  Also,  the  feces  of  healthy  animals  (sheep)  that 
have  grazed  upon  anthrax-fields  contain  spores  under  cer- 
tain conditions.  The  introduction  of  anthrax -bacilli  into 
the  gastro-intestinal  tract  does  not  invariably  induce 
anthrax.  The  spores  in  some  cases  pass  through  the 
digestive  tract  without  causing  injury,  although  they  remain 
as  infective  for  other  animals  as  they  were  formerly.  Also, 
this  mode  of  infection  presupposes  the  existence  of  an 
injury  or  a  lesion  of  the  mucous  membrane.  With  hay 
obtained  from  anthrax-fields  the  spores  gain  entrance  into 
stables  and  give  rise  to  so-called  stable -epidemics.  As 
a  result  of  floods  in  the  infected  districts,  the  germs  are 
carried  far  and  wide,  and  thus  give  rise  to  cases  of  anthrax 
at  places  where  previously  the  disease  was  unknown. 

In  human  beings  gastro-intestinal  anthrax  is  by  no  means 
so  common  as  malignant  pustule  ;  it  was  described  by 
earlier  observers  as  intestinal  mycosis.  Probably  it  occurs 
more  commonly  than  current  reports  would  indicate.  The 
cases  pursue  the  clinical  course  of  dysentery  or  of  typhoid 
fever,  and  if  bacteriologic  examination  is  omitted  at  the 
autopsy,  they  may  readily  escape  diagnosis. 

ic)  Lungs.- — Pulmonary  anthrax  has  been  repeatedly 
observed  in  England  (Bradford)  in  individuals  who  pull 
sheep's  wool  or  work  with  goat's  or  camel's  hair  and  the 
like  {wool-sorter  s  disease^,  and  in  Germany  in  those  who  as- 
sort rags  (ragpicker' s  disease^. 

Experimental  Development  of  Anthrax. — All  of  the 
modes  of  infection  that  have  been  mentioned  as  possible  in 
accordance  with  clinical  experience  may  be  imitated  in  ex- 
periments on  animals.  Susceptible  animals  die  of  anthrax- 
septicemia  after  cutaneous  and  subcutaneous  inoculation. 
The  spleen  in  such  animals  is  quite  considerably  enlarged 
at  autopsy,  its  consistency  is  soft  and  friable,  its  color  dark. 
If  spore-containing  material  is  fed  to  sheep,  four  among  ten 
animals,  on  an  average,  die  after  the  introduction  of  small 
amounts  ;  if  large  amounts  are  used,  almost  all  die.  The 
spores,  thus,  escape  uninjured  the  influence  of  the  acid  gas- 
tric juice.  Anthrax  occurs  but  seldom  in  rabbits,  guinea- 
pigs,  and  white  mice  as  a  result  of  feeding-experiments. 

Buchner  has  made  careful  observations  with  regard  to  the 
occurrence  of  pulmonary  anthrax.  When  the  spores  are 
inhaled  experimentally,  the  animals  die  of  general  infection. 


296  CLINICAL  BACTERIOLOGY. 

The  permanent  forms  penetrate  the  intact  mucous  mem- 
brane of  the  alveoli,  and  gain  entrance  into  the  lymphatics 
and  blood-vessels,  in  which  they  germinate.  After  inhala- 
tion of  anthrax-bacilli,  however,  such  migration  does  not 
take  place.  The  bacilli  remain  inactive,  and  cause  only 
local  irritation — circumscribed  inflammation  of  the  lungs. 
In  this  connection  it  may  be  noted  that  in  quite  rare  cases 
of  anthrax  the  disease  has  also  in  human  beings  pursued 
the  clinical  course  of  septicemia-^that  is,  without  local  in- 
fection and  without  malignant  pustule.  The  portal  of 
infection  remained  undiscovered  in  these  cases,  although  it 
is  possible  that  this  part  was  taken  by  the  lungs. 

Distribution  of  Anthrax-bacilli  in  the  Infected  Body. 
— In  cases  of  general  infection  with  anthrax  the  bacilli  are 
found  in  the  blood,  and  in  only  a  small  number  of  instances 
in  the  larger  vessels,  the  greater  amount  being  present  in  the 
capillary  area.  The  capillaries  of  all  the  organs,  especially 
the  spleen  and  the  liver,  are  filled  by  the  bacilli,  sometimes 
actually  packed  with  them,  so  that  their  lumen  appears  to 
be  occluded.  Most  of  the  bacilli  lie  with  their  long  axes 
parallel  to  the  walls  of  the  vessel — that  is,  they  are  directed 
in  the  course  of  the  blood-stream.  In  the  glomeruli  of  the 
kidneys  and  in  the  intestinal  villi  the  mass  of  bacteria  fre- 
quently induces  capillary  hemorrhages.  The  microorgan- 
isms in  this  way  gain  entrance  into  the  uriniferous  tubules, 
although  generally  they  do  not  progress  beyond  the  con- 
♦voluted  tubes  (Koch).  Anthrax  in  animals  may,  therefore, 
be  considered  as  the  type  or  paradigm  of  a  true  infection,  the 
factor  of  intoxication  remaining  entirely  in  the  background 
and  the  most  prominent  feature  being  the  enormous  multi- 
plication of  the  exciting  agents.  On  examining  the  viscera 
of  animals  dead  of  anthrax  the  impression  is  gained  that 
death  has  resulted  from  a  flooding  of  the  capillary  area  with 
the  bacilli.  The  anthrax-bacilli  are,  however,  by  no  means 
found  in  the  blood  immediately  after  infection,  but  several 
hours  always  elapse  before  they  appear.  The  animal,  how- 
ever, will  previously  have  been  sick.  This  indicates  that  the 
formation  of  toxin  on  the  part  of  the  bacteria  is  not  en- 
tirely wanting — a  circumstance  whose  certainty  is  estab- 
lished by  the  course  of  the  disease  in  less  susceptible  ani- 
mals (and  in  human  beings),  in  which  the  lesion  remains 
localized. 

In  cases  of  malignant  pustule  the  bacilli  are  present  in 


ANTHRAX.  297 

considerable  number  only  before  the  wall  of  leukocytes 
that  separates  the  eschar  from  the  subjacent  tissues.  They 
surround  the  hair-follicles  and  the  sweat-glands,  but  their 
distribution  bears  no  relation  to  the  blood-vessels. 

The  infiltrates  in  cases  of  intestinal  anthrax  are,  both 
histologically  and  bacteriologically,  to  be  placed  upon  the 
same  plane  as  malignant  pustule.  The  mesenteric  glands 
are  swollen  and  filled  with  the  parasites. 

In  cases  of  pulmonary  anthrax  if  a  local  lesion  has 
developed,  the  bacilli  are  found  in  the  perivascular  lymph- 
spaces.  If  a  lesion  is  wanting  at  the  portal  of  entry,  the 
swollen  bronchial  glands  nevertheless  constantly  contain 
the  bacilli. 

Death  occurs  in  every  variety  of  anthrax,  usually  as  a 
result  of  general  infection.  In  addition  to  the  blood  the 
parasites  pass  over  into  the  milk,  the  bile,  the  saliva,  and 
the  feces.     They  are  found  less  commonly  in  the  urine. 

Toxins  of  Anthrax-bacilli.  —  Practically  nothing  is 
known  with  regard  to  the  metabolic  products  of  the  anthrax- 
bacillus.  Hankin  obtained  a  toxic  albumose  from  pure 
cultures.  With  regard  to  the  relation  between  this  albumin- 
ous substance  and  the  actual  anthrax-toxin,  the  same  state- 
ment may  be  made  as  that  with  regard  to  the  toxalbumins 
of  Brieger  and  Frankel  (pp.  29,  30). 

Mixed  Infection. — In  cases  of  anthrax  mixed  infection 
is  not  without  significance.  The  limiting  suppuration, 
which  brings  about  the  exfoliation  of  the  malignant  pus- 
tule or  of  the  intestinal  filtrate,  may  be  excessive  and  give 
rise  to  phlegmons  and  to  septicemia  or  pyemia  ;  many  a  pa- 
tient who  has  survived  the  anthrax-infection  proper  has  died 
later  of  these  secondary  suppurative  processes.  From  the 
blood  and  from  the  internal  viscera  staphylococci  and 
streptococci  may  then  be  cultivated. 

Heredity. — In  the  case  of  anthrax  it  has  been  demon- 
strated experimentally  that  the  bacillus  passes  from  the 
mother  to  the  fetus  only  when  changes  have  taken  place  in 
the  placenta — minimal  hemorrhages  will  suffice.  Intrauter- 
ine infection  with  anthrax  has  also  been  observed  in  human 
beings  (Marchand).  As  bearing  upon  the  question  whether 
the  fetus  in  utero  may  infect  its  mother,  the  experiments  of 
Lingard  are  interesting.  This  observer  infected  rabbit- 
fetuses  in  utero  with  anthrax.  As  a  rule,  the  mothers  were 
not  infected,  but  they  later  proved  immune  to  the  disease. 


298  CLINICAL  BACTERIOLOGY. 

the  immunity  lasting  for  more  than  eight  months.  In  this 
way  would  be  demonstrated  the  law  for  anthrax  that  CoUes 
propounded  for  syphilis  (p.  318). 

Bacteriologic  Diagnosis. — From  the  deeper  parts  of  a 
suspected  pustule  some  tissue-fluid  is  removed  and  plates 
are  made  therefrom.  If  suspicious  colonies  develop,  pure 
cultures  are  made  and  animals  infected  therewith.  If  intes- 
tinal anthrax  is  suspected,  the  vomited  matters  and  the  feces 
should  be  examined  bacteriologically.  In  cases  of  pulmon- 
ary anthrax  the  abundant  frothy  sputum  at  times  contains 
the  bacilli.  Examination  of  the  blood  will  yield  informa- 
tion as  to  whether  general  infection  with  anthrax  is  present 
or  not.  Such  examination  is,  therefore,  always  of  the 
greatest  importance  in  the  formulation  of  the  prognosis.  It 
must,  however,  always  be  borne  in  mind  that  the  bacilli  are 
present  especially  in  the  capillary  area. 

For  sanitary  purposes  it  is  frequently  necessary  to  decide 
whether  an  animal  has  died  of  anthrax  or  not.  If  death 
has  taken  place  but  a  short  time  previously,  microscopic 
examination  of  fluid  from  the  spleen  and  of  the  blood  will 
suffice.  The  presence  of  the  bacilli  with  capsules,  demon- 
strable by  the  method  of  Johne  (p.  288),  renders  the  diag- 
nosis positive.  If,  however,  one  or  more  days  have  elapsed 
since  the  death  of  the  animal,  then  there  develop  in  the  body 
of  animals  dead  of  anthrax  cadaver-bacilli  that  can  be  dif- 
ferentiated from  the  specific  exciting  agents  of  anthrax  only 
with  exceeding  difficulty.  Under  such  conditions  gelatin- 
plates  must  be  made,  and  mice  and  guinea-pigs  inoculated 
with  fluid  from  the  spleen. 

In  inoculating  the  animals  the  possibility  of  the  presence 
of  the  bacillus  of  malignant  edema  must  be  borne  in 
mind,  as  this  microorganism  likewise  is  not  rarely  found 
in  the  bodies  of  dead  animals.  On  subcutaneous  in- 
oculation the  latter  predominates,  and  the  animals  die  of 
malignant  edema  notwithstanding  the  presence  of  anthrax- 
bacilli.  This  source  of  error  can  be  avoided  by  inoculat- 
ing the  animals  cutaneously  with  the  suspected  material. 
The  anthrax-bacillus  alone  will  then  develop,  and  the 
diagnosis  can  not  go  astray.  If  putrefaction  has  pro- 
gressed too  far  in  the  body  to  be  examined,  then  the 
anthrax-bacilli  may  be  completely  suppressed  by  the  com- 
petition of  the  other  varieties  of  bacteria,  so  that  the  diag- 
nosis is  no  longer  possible. 


ANTHRAX.  299 

Immunity  and  Vaccination, — Recovery  from  malignant 
pustule  does  not  confer  immunity,  at  least  none  of  any 
duration.  It  has  been  repeatedly  observed  that  the  same 
individual  has  been  attacked  two  or  three  times  with 
malignant  pustule,  and  even  at  intervals  of  a  few  months. 
The  second  attack  often  pursues  even  a  more  virulent 
course  than  the  first.  Nevertheless,  it  is  possible  experi- 
mentally to  immunize  animals  to  anthrax.  The  anthrax- 
bacillus  is  the  microbe  with  which  Pasteur  demonstrated 
for  the  first  time  that  the  action  of  heat  diminishes  the 
virulence  materially.  Pasteur  cultivated  anthrax-bacilli  at 
a  temperature  of  42°  C.  (107.6°  F.).  They  still  developed 
at  this  temperature,  but  gradually  suffered  in  virulence  from 
day  to  day.  If  after  the  lapse  of  several  days  the  bacilli 
thus  attenuated  are  transferred  to  a  new  nutrient  medium 
and  are  exposed  to  a  more  favorable  temperature  (35°  C. — 
95°  F.),they  then  grow  luxuriantly  again,  but  their  virulence 
remains  attenuated  ;  the  bacilli  do  not  recover.  Upon  the 
basis  of  these  fundamental  facts  Pasteur  prepared  two 
varieties  of  attenuated  anthrax-bacilli,  one  of  which  had 
been  exposed  for  from  fifteen  to  twenty  days  to  a  tempera- 
ture of  42°  C.  (107.6°  F.),  and  which  was  capable  of 
destroying  only  guinea-pigs  not  more  than  twenty-six  hours 
old  (Vaccine  I).  The  other  had  been  exposed  from  ten  to 
twelve  days  to  a  temperature  of  42°  C.  (107.6°  F.),  and  it 
was  capable  of  destroying  guinea-pigs,  white  mice,  and 
rarely  rabbits  (Vaccine  II). 

According  to  the  method  of  Pasteur,  animals  (rabbits, 
sheep,  cows)  are  inoculated  first  with  Vaccine  I.  They  be- 
come sick  and  exhibit  fever  of  greater  or  less  intensity. 
After  the  disease-symptoms  have  disappeared,  the  animals 
are  inoculated  with  Vaccine  II,  and  the  same  symptoms 
are  repeated.  After  the  animals  have  recovered  from  this 
second  disturbance  they  are  thoroughly  protected  against 
inoculation  with  highly  toxic  anthrax-material. 

The  attenuated  anthrax-cultures  generate  alkaline  sub- 
stances upon  the  nutritive  media ;  whereas  the  virulent 
cultures  generate  a  considerable  amount  of  acid.  The 
vaccines  are  distinguished  from  the  highly  virulent  cultures, 
further,  by  the  fact  that  they  give  rise  to  febrile  movement. 
This  fact  is  usually  interpreted  as  indicating  that  the 
fever  represents  a  reaction  of  the  organism  to  the  invading 
parasites.     As  a  matter  of  fact,  it  has  for  a  long  time  been 


300  CLINICAL   BACTERIOLOGY. 

a  prognostic  rule  clinically,  peculiarly  in  the  case  of  anthrax, 
that  complete  apyrexia  is  a  sign  of  ill  omen. 

Pasteur's  method  of  vaccination  confers  complete  and  se- 
cure protection  against  subcutaneous  anthrax.  As  to  its 
protective  power  with  regard  to  field-anthrax,  opinions  have 
differed.  Koch,  Lofifler,  and  Gaffky  express  themselves 
with  some  reserve  in  this  connection.  They  do  not  believe 
that  the  protective  inoculation  confers  absolute  immunity  to 
intestinal  anthrax.  Statistics,  however,  appear  unquestion- 
ably to  favor  the  view  of  Pasteur.  Since  the  practice  of 
vaccinating  the  herds  has  been  instituted  in  France,  the 
mortality  from  anthrax  among  grazing  cattle  has  been 
materially  reduced. 

The  blood-serum  of  animals  immunized  to  anthrax  does 
not,  so  far  as  is  known,  transmit  immunizing  properties  to 
other  animals. 

Prophylaxis. — The  prophylaxis  against  anthrax  consists 
in  rendering  innocuous  the  bodies  of  animals  and  human 
beings  dead  from  the  disease.  This  end  is  attained  in  the 
simplest  manner  by  incinerating  the  bodies  as  a  whole. 
The  danger  of  infection,  however,  is  maintained  by  the 
introduction  and  manipulation  of  suspicious  material  (hair, 
wool,  rags,  leather)  from  foreign  countries  in  which  the 
sanitary  precautions  observed  by  most  intelligent  nations 
are  not  enforced.  In  England,  the  home  of  wool-sorter's 
disease,  regulations  have  been  put  in  force  requiring  that 
wool,  which  is  derived  principally  from  Asia,  should  be 
boiled  before  it  is  assorted.  Since,  the  disease  has  mate- 
rially diminished.  In  the  same  spirit  the  disinfection  of 
skins,  hair,  wool,  etc.,  of  suspicious  origin  should  be 
everywhere  insisted  upon  before  being  manipulated. 


GLANDERS. 

The  exciting  agent  of  glanders  was  discovered  in  1882 
by  Lofifler  and  Schiitz. 

Morphology  of  the  Glanders-bacilli. — The  glanders- 
bacilli  are  small,  slender,  nonmotile  rods,  at  times  curved,  with 
rounded  extremities,  from  2  to  3  ^t  long,  and  from  0.2  to 
0.4/ji  thick.  As  a  rule,  they  lie  singly.  The  question  whether 
glanders-bacilli  form  spores  is  still  an  open  one,  though  answered 
in  the  affirmative  by  Baumgarten  and  Rosenthal,  who  succeeded 
in  staining  spores,  and  in  the  negative  by  most  observers,  because 


GLANDERS.  301 

the  germination  of  the  so-called  spores  has  not  yet  been  ob- 
served. 

The  glanders-bacillus  takes  all  stains  without  difficulty,  but  it 
is  decolorized  with  equal  facility.  The  best  specimens  are  ob- 
tained in  cover-slip  preparations  by  treatment  with  hot  Loffler's 
solution  or  hot  carbolfuchsin,  and  decolorization  with  distilled 
water.  The  organisms  do  not  stain  by  Gram's  method.  In 
stained  preparations  the  bacilli  exhibit  almost  regularly  un- 
stained deficiencies.  The  bacilli  frequently  appear  in  the  form 
of  short  structures  resembling  cocci.  They  are  facultative 
anaerobic.  The  temperature-minimum  is  25°  C.  (77°  F. )  ;  the 
temperature-optimum,  37°  C.  (98.6°  F.)  or  38°  C.  (100.4° 
F.)  j  the  temperature-maximum,  42°  C.  (107.6°  F.). 


i 


■'.'S-    r-^^' 


'V;-/nSpii^ 


Fig.  65. — Bacillus  mallei,  from  a  culture  upon  glycerin  agar-agar;   X  1000  (Frankel 

and  Pfeiffer). 

Cultural  Properties. — On  glycerin-agar  plates  glistening, 
granular  colonies,  with  a  yellowish  tinge  and  a  smooth  border, 
appear. 

Streak- cultures  upon  Glycerin-agar. — Along  the  line  of  inocu- 
lation a  moist,  whitish  coating  forms.  Upon  blood-serum  iso- 
lated, translucent,  yellowish  drops  appear,  which  do  not  liquefy 
the  culture-medium.  Upon  gelatin  slight  growth  takes  place  at 
25°  C.  (77°  F.),  with  slight  liquefaction.  Bouillon  is  rendered 
densely  turbid.  The  growth  of  glanders-bacilli  upon  potato 
is  characteristic.  The  inoculated  surface  exhibits  after  two  days 
a  thin,  honey-yellow  deposit,  which  after  a  week  becomes  quite 
dark,  brownish-red,  and  surrounded  by  a  slightly  blue,  irides- 
cent zone.     The  best  culture-medium  is  glycerin-agar. 


302  CLINICAL  BACTERIOLOGY. 

Resistance  of  Glanders-bacilli. — For  the  continued 
cultivation  of  glanders-bacilli  it  is  important  to  know  that 
the  organisms  rapidly  undergo  natural  attenuation  as  early 
as  the  fourth  or  fifth  generation.  If,  therefore,  it  is  desired 
to  obtain  virulent  bacilli,  it  is  necessary  to  interpolate  inocu- 
lations of  animals  every  two  or  three  culture-generations. 

In  the  dry  state  the  glanders-bacilli,  according  to  Lofifler, 
retain  their  vitality  for  three  months.  Other  observers, 
however,  have  found  them  dead  within  ten  days  when  dried 
in  a  thin  layer.  Toward  heat  the  glanders-bacilli  prove 
quite  resistant.  They  are  destroyed  in  two  minutes  at  a 
temperature  of  ioo°  C.  (212°  F.)  ;  in  five  minutes  at  a 
temperature  of  80°  C.  (176°  F.)  ;  in  fifteen  minutes  by 
I  :  1000  mercuric  chlorid  ;  and  in  an  hour  by  five  per 
cent,  carbolic  acid. 

Susceptibility  of  Animals  to  Glanders. — Among  do- 
mestic animals  there  are  susceptible  in  diminishing  degree 
asses,  mules,  horses,  goats,  cats,  sheep,  dogs,  swine. 
Cattle  are  immune.  Among  experimental  animals  field- 
mice,  wood-mice,  and  guinea-pigs  exhibit  the  most  pro- 
nounced, and  rabbits  a  much  slighter,  predisposition. 
White  mice  and  domestic  mice  prove  entirely  insusceptible 
to  glanders.  If,  however,  white  mice  are  previously  pois- 
oned with  phloridzin,  they  succumb  to  infection  with 
glanders  (Leo).  Birds,  with  the  exception  of  pigeons,  are 
refractory  to  glanders.  In  all  animals  glanders  is  at  first 
a  local  disease,  but  later  it  becomes  generalized  and  attacks 
all  of  the  viscera. 

Occurrence  and  Distribution  of  the  Bacilli  in  the 
Products  of  the  Disease. — From  a  pathologic-anatomic 
standpoint  glanders  belongs  to  the  group  of  diseases  that, 
like  tuberculosis,  give  rise  to  the  formation  of  nodules 
that  exhibit  a  marked  tendency  to  disintegration  and 
softening.  The  small,  often  only  submiliary,  grayish  new- 
formations  consist  of  epithelioid  cells  and  mainly  of  leuko- 
cytes. The  glanders-bacilli  are  present  especially  at  the 
center  of  the  granulations.  They  are  demonstrable  with 
difficulty  by  staining.  The  best  mode  of  procedure  con- 
sists in  placing  the  sections  for  from  six  to  eight  hours  in 
carbol-methylene-blue  (methylene-blue  i,  absolute  alcohol 
10  cu.  cm.,  carbolic  acid,  5  per  cent.,  100  cu.  cm.)  or  car- 
bolfuchsin,  next  decolorizing  in  dilute  acetic  acid,  and  then 
in  distilled  water,  drying  upon  a  slide,  and,  after  clearing  in 


GLANDERS.  303 

xylol,  mounting  the  preparation  in  xylol  Canada  balsam. 
The  sections  may  also  be  treated  according  to  Weigert's 
method  (p.  io6),  Loffler's  alkaline  methylene-blue  solution 
being  selected  for  staining.  In  this  way  positive  results 
are  obtained  only  with  the  relatively  recent  nodules.  If 
necrosis  has  taken  place,  the  bacilli  are  only  rarely  found 
in  the  products  of  disintegration. 

Experiments  on  Animals. — Small  numbers  of  bacilli 
inoculated  subcutaneously  into  susceptible  animals  (gener- 
ally guinea-pigs  or  field-mice),  or  larger  numbers  rubbed 
into  the  uninjured  skin,  cause  death  with  considerable  cer- 
tainty. The  mice  die  quickly — within  three  or  four  days — 
and  the  spleen,  the  liver,  and  the  lungs  are  filled  with  an 
enormous  number  of  nodules  scarcely  visible  to  the  naked 
eye.  Guinea-pigs  are  better  adapted  for  the  study  of  the 
course  of  infection.  In  them  a  local  infection  first  develops, 
an  infiltration,  that  soon  is  converted  into  an  ulcer  with 
indurated  margins.  Then  follow  swelling  and  suppuration 
of  the  adjacent  lymphatics  and  lymph-glands,  and  finally 
general  infection  with  the  characteristic  new-formations. 
The  process  advances  by  way  of  the  lymph-paths.  Apart 
from  cases  pursuing  a  very  acute  course,  the  blood  almost 
never  contains  the  bacteria.  The  infection  of  the  lymphatic 
apparatus  extends  with  exceeding  rapidity.  As  early  as 
an  hour  after  inoculation  of  a  superficial  skin- wound  cauter- 
ization of  the  latter  will  no  longer  suffice  to  prevent  the 
development  of  the  disease.  The  urine,  the  seminal  fluid, 
the  sweat,  the  saliva,  and  the  aqueous  humor  of  infected 
animals  may  contain  the  parasites.  The  spleen  and  the 
bile  are  said  to  be  free  from  them. 

Portals  of  Entry  For  and  Course  of  Glanders. — In 
human  beings,  who,  in  the  vast  majority  of  cases,  are  infected 
by  contact  with  horses  suffering  from  glanders,  the  skin,  with- 
out doubt,  constitutes  the  principal  portal  of  infection.  The 
individuals  in  question,  usually  hostlers,  coachmen,  farmers, 
cavalrymen,  and  the  like,  may  be  infected  through  the  in- 
termediation of  the  most  superficial  and  most  insignificant 
wounds  of  the  skin.  Laboratory-infection  with  glanders, 
in  manipulating  infective  material  and  glanders-bacilli, 
has  been  observed  repeatedly.  Infection  sometimes  takes 
place  also  through  the  mucous  membranes.  Cases  are  on 
record  in  which  hostlers  have  acquired  the  disease  by 
drinking  from  the  same  pail  as  their  sick  horses,  and  the 


304  CLINICAL  BACTERIOLOGY. 

like.  Whether  or  not  infection  may  take  place  by  way  of 
the  respiratory  apparatus  has  not  yet  been  decided  with 
certainty.  Nevertheless,  it  is  remarkable  that  in  horses 
glanders,  -in  its  initial  stage,  is  seated  in  the  nasal  cavity 
(glanders-ulcer).  Bollinger  believes  that,  especially  in  those 
attacked  by  glanders  who  present  general  symptoms  in 
advance  of  the  local  manifestations,  the  virus  has  gained 
entrance  into  the  body  through  the  respiratory  passages. 
The  use  of  meat  from  animals  suffering  from  glanders  may, 
likewise,  give  rise  to  the  disease.  At  least  a  number  of 
feeding-experiments  in  animals  are  in  favor  of  this  view. 
Such  experiments  have  yielded  positive  results  in  cats,  dogs, 
lions,  and  bears.  On  the  other  hand,  other  experiments 
with  the  feeding  of  glanderS-material  have  yielded  negative 
results.  The  muscles  themselves  do  not  harbor  the  para- 
sites, but  rather  the  lymphatic  vessels  and  glands  lying 
within  them  or  near  them. 

TJie  clinical  picture  of  glanders  in  human  beings  is  a  rather 
variable  one,  in  accordance  with  the  site  of  infection.  Usu- 
ally there  occurs  locally  at  the  portal  of  infection  a  swelling, 
and  this  is  soon  followed  by  tumefaction  and  suppuration  of 
the  neighboring  lymphatics.  Then  multiple  abscesses  form 
in  the  skin,  the  muscles,  and  the  internal  viscera,  and  often 
suppuration  takes  place  in  joints.  The  clinical  picture  re- 
sembles that  of  pyemia.  Upon  the  mucous  membranes, 
and  particularly  in  the  nose,  characteristic  glanders-nodules 
appear,  which  soon  disintegrate  and  give  rise  to  ulcers. 
Death  results  from  the  general  infection,  which  takes  place 
in  human  beings  by  way  of  the  lymphatics.  An  acute  and 
a  chronic  variety  of  glanders  have  been  observed.  In  the 
former  suppuration  is  the  more  likely  to  occur ;  in  the 
latter — so-called  farcy — tissue-proliferation  is  the  more 
conspicuous. 

Heredity. — The  transmission  of  glanders-bacilli  from  the 
mother  to  the  fetus  has  been  observed  repeatedly.  The 
following  observation  by  Loffler  is  interesting.  A  female 
guinea-pig  that  had  recovered  from  inoculation  with  glan- 
ders gave  birth  to  offspring  five  months  later.  Apparently 
healthy  at  birth,  the  young  animal  died  of  visceral  glanders 
at  the  age  of  a  week. 

Bacteriologic  Diagnosis  of  Glanders. — The  bacterio- 
logic  diagnosis  has  to  contend  with  the  difficulty  that  in  the 
course  of  suppurative  destruction  in  the  foci  of  disease  the 


MALIGNANT   EDEMA.  305 

bacilli  also  are  destroyed.  In  suspicious  cases,  however, 
agar-plates  should  always  be  made  with  the  pus  from  the 
ulcers  in  question.  In  addition  male  guinea-pigs  should 
receive  intraperitoneal  injections  of  the  suspected  secretion. 
If  the  disease  be  glanders,  swelling  of  the  testicles  takes 
place  in  the  course  of  two  or  three  days.  This  is  absolutely 
characteristic,  and  is  subsequently  followed  by  suppuration 
of  the  organs  (Straus).  The  injection  must  always  be  made 
in  the  median  line,  as  otherwise  there  is  danger  of  injuring 
the  seminal  vesicles,  and  as  by  direct  injection  into  these 
structures  other  microorganisms  also  may  give  rise  to 
swelling  and  suppuration  of  the  testicles.  . 

Mallein. — In  exactly  the  same  manner  as  tuberculin  is 
prepared,  von  Preusse  and  Kalning  obtained  from  cultures 
of  glanders-bacilli  a  lymph — mallein — representing  a  proteid 
substance  of  the  glanders-bacilli.  Mallein  is  employed  in 
veterinary  medicine  for  diagnostic  purposes.  Horses  suf- 
fering from  glanders  react  to  injection  of  the  lymph  with 
fever. 

Prophylaxis. — The  most  efficient  prophylaxis  against 
glanders  consists  in  the  destruction  of  animals  suffering 
from  the  disease,  and  the  incineration  of  their  carcases. 
The  remaining  horses  in  a  stable  in  which  the  disease  has 
appeared  must  be  subjected  to  rigid  quarantine,  and  the 
attendants  must  have  their  attention  called  to  the  danger 
of  infection  and  be  enjoined  to  careful  disinfection. 


MALIGNANT  EDEMA. 

The  exciting  agent  of  malignant  edema,  which  was  de- 
scribed in  1 88 1  by  Koch,  is  identical  with  the  vibrion  sep- 
tique  of  Pasteur. 

The  bacillus  of  malignant  edema  (vibrio  septicus)  is  a 
slender  bacillus  about  as  long  as  the  anthrax-bacillus,  but  some- 
what narrower  (from  o.S.to  i  //),  with  rounded  extremities.  It 
possesses  spontaneous  motility,  which  is  due  to  from  three  to 
twelve  lateral  flagella.  The  vibrio  septicus  has  a  tendency  to 
form  long  filaments,  both  in  culture  and  also — in  contradis- 
tinction from  the  anthrax-bacillus — in  still  greater  degree  in 
the  living  body.  The  chains  not  rarely  present  pretty,  arch- 
like curves.  The  temperature-optimum  is  37°  C.  (98.6°  F.), 
although  the  bacillus  will  grow  at  room -temperature.     Above 


306 


CLINICAL  BACTERIOLOGY. 


20°  C.  (68°  F. )  it  is  capable  of  sporulation.  The  spore,  situ- 
ated centrally,  is  at  times  wider  than  the  bacillus,  which  then 
assumes  a  spindle-shaped,  distended  appearance.  The  vibrio 
septicus  is  a  rigidly  anaerobic  organism.  It  stains  without 
difficulty  with  all  of  the  aniline  dyes,  but  not  by  Gram's 
method. 

Cultural  Properties. — O^  gelatin-plates  the  colonies  present 
the  appearance  of  glistening,  hollow  globules,  filled  with  fluid. 
At  their  center,  with  a  magnification  of  from  80  to  100,  a  dense 
network  of  intimately  intertwined  threads  may  be  seen,  with 
radiating  processes  at  the  periphery.  On  more  careful  exami- 
nation it  will  be  observed  that  the  colony  is  in  motion. 


Fig.  66. — Bacillus  of  malignant  edema,  from  the  body-juice  of  a  guinea-pig  inoculated 
with  garden-earth;  X  looo  (Frankel  and  Pfeiffer). 


On  agar-plates  small,  irregular,  whitish,  translucent  colonies 
appear,  with  a  dense  center,  from  which  innumerable  delicate 
ramifications  proceed. 

In  high  gelatin  stab- culture  liquefaction  and  turbidity  of  the 
underlying  gelatin  take  place,  with  abundant  formation  of  gas, 
especially  on  addition  of  reducing  substances. 

In  high  agar  stab-cultures  the  line  of  inoculation  exhibits  ser- 
rated branched  margins.     Abundant  gas-production  takes  place. 

Bouillon  is  rendered  turbid,  and  later  a  precipitate  forms. 
The  reaction  is  not  changed,  but  carbon  dioxid  and  hydrogen 
develop. 

All  cultures  generate  a  disagreeable,  fetid  odor. 


MALIGNANT   EDEMA.  307 

Experiments  on  Animals. — Horses,  swine,  sheep,  goats, 
dogs,  pigeons,  ducks,  hens,  rabbits,  mice,  and  guinea-pigs 
are  susceptible  to  mahgnant  edema.  In  guinea-pigs  sub- 
acute infection  pursues  quite  a  characteristic  course.  The 
animals  cower,  their  hair  stands  erect,  and  signs  of  great 
fear  set  in.  On  the  slightest  touch  the  animals  cry  out. 
Death  takes  place  within  twelve  hours.  On  autopsy 
there  is  found  at  the  site  of  inoculation  quite  con- 
siderable bloody  edema,  with  a  small  number  of  gas- 
bubbles,  involving  the  subcutaneous  connective  tissue  and 
the  superficial  muscles.  In  this  area  the  bacilli  with 
spores  are  found  in  large  numbers.  On  the  other  hand, 
the  blood  and  the  internal  viscera  are  always  free  from 
bacteria  if  the  autopsy  is  performed  immediately  after 
death.  As  long  as  the  animal  continues  to  live,  the  anae- 
robic edema-bacilli  are  not  capable  of  multiplying  in  the 
oxygen-containing  blood.  Only  after  death  has  taken 
place  do  they  advance,  and  soon  the  viscera  and  the  blood 
are  filled  with  them.  The  spleen  of  the  animal  is  large 
and  diffluent.  The  liver  and  the  lungs  are  pale,  and  the 
latter  are  of  a  peculiar  grayish-red  color.  In  mice  the 
bacilli  gain  entrance  into  the  blood  and  the  internal  viscera 
during  life. 

In  the  main,  malignant  edema  represents  an  intoxication. 
As  a  matter  of  fact,  precisely  the  same  clinical  picture  can 
be  developed  experimentally  in  animals  if  the  mature 
bouillon-culture  or  the  serum  from  the  edema  freed  of  bac- 
teria by  passage  through  a  porcelain  filter  is  injected  intra- 
peritoneally  into  guinea-pigs  in  rather  considerable  amount. 

Occurrence  of  the  Vibrio  Septicus. — The  bacilli  of 
malignant  edema,  or  rather  their  resistant  permanent  forms, 
are  widely  distributed  in  nature.  They  are  the  attendants 
of  putrefactive  processes,  especially  those  that  take  place  in 
the  absence  of  oxygen.  They  are  found  present  in  manure, 
in  dust,  and  in  the  earth  of  gardens  and  of  fields.  If  a 
small  amount  of  earth  be  introduced  into  a  pocket  beneath 
the  skin  in  a  guinea-pig  or  a  rabbit,  the  animal  will  die  of 
malignant  edema.  The  picture  developed  under  these  con- 
ditions is,  however,  not  a  pure  one.  In  addition  to  the 
septic  vibrios  a  large  number  of  other  microbes  will  be 
found  present  in  the  edema,  and  as  a  result  of  this  mixed 
infection  the  exudate  is  not  merely  bloody  and  serous,  but 
putrid  and  fetid. 


308  CLINICAL  BACTERIOLOGY. 

Malignant  Edema  in  Human  Beings. — Malignant 
edema  occurs  at  present  but  seldom  in  human  pathology.  In 
preantiseptic  times  it  was  more  common  and  more  alarming. 
The  so-called  septicemie  gangreneuse  and  the  gangrene 
gazeuse  of  the  French  were  in  part  manifestations  of  the  ac- 
tivity of  the  vibrio  septicus,  and  identical  with  our  malig- 
nant edema.  In  order  that  the  bacillus  of  malignant  edema 
may  exhibit  its  activity,  the  wounds  through  which  infection 
takes  place  must  be  deep,  because  the  strictly  anaerobic 
parasite  has  no  opportunity  for  development  upon  the  sur- 
face. The  bacillus  may,  further,  be  associated  in  mixed  infec- 
tion with  other  microorganisms,  which  consume  the  oxygen 
available,  and  thus  artificially  produce  an  atmosphere  free 
from  this  gas.  It  may  be  mentioned  that  in  two  patients 
suffering  from  typhoid  fever  malignant  edema  has  been  ob- 
served after  subcutaneous  injection  of  tincture  of  musk. 

The  portal  of  infection  is  always  constituted  by  a  breach 
in  continuity  of  the  external  integument. 

Bacteriologic  Diagnosis. — Plates  are  made  from  the 
putrid  exudate  in  an  atmosphere  of  hydrogen,  and  at  the 
same  time  guinea-pigs  are  inoculated  subcutaneously. 

Immunity. — Malignant  edema  is  one  of  those  bacterial 
diseases  in  which  artificial  immunization  through  metabolic 
products  was  first  effected.  Chamberland  and  Roux  im- 
munized guinea-pigs  by  means  of  intraperitoneal  injections 
of  bouillon-cultures  sterilized  by  exposure  for  ten  minutes 
in  the  autoclave  at  a  temperature  of  between  105°  C. 
(221°  F.)  and  110°  C.  (230°  F.).  Immunity  to  malignant 
edema  is  also  established  without  difficulty  in  animals  by  all 
other  methods. 


PROTEUS-INFECTIONS. 

Morphology  of  the  Proteus. — The  proteus-bacteria,  dis- 
covered by  Hauser  in  1885,  are  small,  motile  rods  of  ex- 
ceeding activity  and  variable  size. 

The  proteus-bacteria  are  usually  arranged  in  pairs,  and  not 
rarely  also  in  longer  filaments.  In  addition  to  these  funda- 
mental forms  coccus-like  bodies  and  long,  winding  threads  (spir- 
ulins)  also  are  encountered.  The  proteus  is  characterized  by 
the  possession  of  an  unusually  large  number  of  flagella  surround- 
ing the  body  of  the  cell.  It  is  stained  readily  with  carbolfuch- 
sin,  and  less  well  with  watery  solutions  of  aniline  dyes.     It  does 


PROTEUS-INFECTIONS.  309 

not  stain  by  Gram's  method.  The  proteus  thrives  equally  well 
at  room-temperature  and  at  the  temperature  of  the  body.  The 
temperature-optimum  is  between  20°  C.  (68°  F.)  and  25°  C. 
(77°  F. ).  The  organism  does  not  give  rise  to  spores,  and  is 
destroyed  by  exposure  for  five  minutes  to  a  temperature  of  55° 
C.  (i3i°F.). 

Cultural  Properties.  —  On  gelatin-plates  small,  round, 
yellowish  colonies  form  at  first,  with  a  dense  center  and  an 
irregular  margin,  from  which  bristle-like  processes  pass  off. 
Other  colonies  are  bounded  by  a  zone  of  filaments  surrounding 
the  central  opaque  mass,  in  part  circularly,  in  part  in  the  most 


/ 


Fig.  67. — Swarming  islands  of  proteus-bacilli  on  the  surface  of  gelatin ;  X  650 
(Hauser). 

varied  loops  and  convolutions.  Extensive  and  rapid  liquefaction 
of  the  gelatin  takes  place.  Into  the  adjacent  nutrient  medium 
extend  processes,  both  straight  and  tortuous,  which  frequently 
are  cut  off  from  the  mother-area  and  move  about  as  free  islands 
in  the  partially  liquefied  gelatin.  These  conditions  may  be  espe- 
cially well  observed  upon  five  or  six  per  cent,  gelatin.  There 
thus  result  peculiar  figures  and  designs,  to  which  the  proteus 
owes  its  name  of  ^*  bacillus  figurans. ' ' 

Gelatine  stab-culture  is  liquefied  with  exceeding  rapidity. 

In  agar  streak-culture  a  grayish,  moist  coating  forms. 

On  potato  a  dirty  grayish  deposit  appears. 

Bouillon  is  rendered  homogeneously  turbid. 


310  CLINICAL  BACTERIOLOGY. 

All  nutrient  media  emit  a  disagreeable  odor. 

Formerly,  Hauser  distinguished  three  distinct  varieties  of 
proteus,  the  proteus  vulgaris,  the  proteus  mirabilis,  and  the  pro- 
teus  Zenkeri ;  but  later  he  abandoned  this  differentiation. 

Experiments  on  Animals. — If  rabbits  or  guinea-pigs 
receive  intraperitoneal  or  intravenous  injections  of  consider- 
able amounts  (three  cubic  centimeters)  of  proteus-culture,  the 
animals  die  of  acute  enteritis  and  peritonitis.  Intravenous 
injection  of  from  five  to  ten  cubic  centimeters  of  a  bouillon- 
culture  is  followed  by  much  more  typical  phenomena  in  the 
dog.  The  animal  suffers  from  bloody  vomiting  and  bloody 
diarrhea  attended  with  severe  tenesmus.  The  temperature 
is  elevated,  and  the  sclerae  are  distinctly  icteric.  On  autopsy 
the  entire  digestive  tract,  from  the  cardia  to  the  anus,  is  the 
seat  of  an  intense  hemorrhagic  inflammation.  The  blood 
and  the  internal  viscera  of  the  dog  contain  none  or  but  a 
few  bacteria.  Exactly  the  same  result  is  obtained  with  fil- 
tered cultures  and  with  the  bodies  of  the  bacilli  carefully 
destroyed.  In  mice,  which,  likewise,  succumb  to  proteus- 
inoculation,  the  bacilli  may  be  cultivated  from  the  viscera. 
The  organisms  become  the  more  virulent  the  oftener  they 
are  passed  through  the  bodies  of  mice. 

Occurrence  of  the  Proteus. — Proteus-bacteria  are 
present  in  all  putrefactive  processes,  and  also  in  the  gastro- 
intestinal canal.  In  human  beings  the  proteus,  in  mixed 
infection  with  the  ordinary  exciting  agents  of  inflammation, 
constitutes  the  cause  o{\h&  putrid,  fetid  phlegmons  at  times 
observed  in  the  sequence  of  cadaveric  infection.  Besides, 
the  proteus  gives  rise  to  so-called  putiid  intoxication,  by 
subsequently  penetrating  into  a  primary  focus  of  suppura- 
tion or  a  traumatic  lesion,  multiplying  there,  and  generating 
metabolic  products  that  are  absorbed.  According  to  H. 
Jager  certain  forms  o{  febrile  icterus,  known  as  Weil's  dis- 
ease, are  caused  by  the  proteus.  Jager  succeeded  in  culti- 
vating a  fluorescent  proteus  from  the  urine,  and  after  death 
also  from  the  viscera  of  individuals  suffering  from  Weil's 
disease.  Infection  had  taken  place  in  these  cases  from 
bathing  in  streams  the  water  of  which  was  contaminated  by 
proteus.  On  the  banks  of  a  tributary  brook  an  epidemic  had 
occurred  among  fowl,  the  exciting  agent  of  which  was  like- 
wise found  to  be  the  proteus  fluorescens.  Further,  the  pro- 
teus was  found  by  E.  Levy  to  be  the  cause  of  hemorrhagic 


GONORRHEA.  311 

gastro-enteritis  from  which  seventeen  persons  suffered  after 
the  use  of  spoiled  meat. 

Bacteriologic  Diagnosis. — Plates  should  be  made  with 
pus  from  the  putrid  phlegmons  and  possibly  also  with  the 
urine  obtained  with  sterile  precautions  from  cases  of  Weil's 
disease. 


GONORRHEA. 

Morphology  of  Gonococci. — The  exciting  agents  of 
gonorrhea,  the  gonococci,  discovered  by  Neisser,  in  1879, 
are  cocci,  which  are  separated  into  two  distinct  hemispheres 
by  means  of  a  dividing  fissure.     The  individual  members 


Fig.  68.— Gonococci  in  urethral  pus;  X  1000  (Frankel  and  PfeifFer). 

suggest  the  appearance  of  a  kidney  or  a  biscuit.  The  cocci 
can  be  stained  with  all  aniline  dyes,  but  not  by  Gram's 
method.  They  are  from  o:"^  to  1.6  /^  long,  and  from  0.6 
to  0.8  /z  thick. 

Occurrence  of  Gonococci. — Gonococci  are  constantly 
present  in  the  secretion  from  all  gonorrheal  inflammations 
(gonorrheal  urethritis,  cervical  catarrh,  blennorrhea,  etc.). 
Their  position  within  the  pus-cells,  in  the  immediate  neigh- 
borhood of  the  nucleus,  is  characteristic.  In  cases  that  are  no 
longer  rare  gonococci  have  been  found  also  in  the  visceral 


312  CLINICAL  BACTERIOLOGY. 

manifestations  of  gonorrhea  (peritonitis,  salpingitis,  oophor- 
itis, endocarditis,  rheumatism,  myelitis).  The  reported 
demonstration  of  gonococci  in  the  normal  urethra — thus 
their  saprophytism — can  not  be  considered  as  established. 

Culture  of  gonococci  (Wertheim)  is  possible  only  at  a 
temperature  of  37°  C.  (98.6°  F.). 

Plate-procedure. — Gonorrheal  pus  is  introduced  into  a  tube 
containing  liquid  human  blood-serum  at  a  temperature  of  40°  C. 
(104°  F.),  and  from  this  two  dilutions  are  prepared  in  two  new 
blood-serum  tubes,  likewise  at  a  temperature  of  40°  C.  (104°  F.). 
Into  each  of  the  three  tubes  a  like  amount  of  liquefied  two  per 
cent,  peptone-agar,  cooled  at  40°  C.  (104°  F.),  is  then  intro- 
duced, thorough  admixture  practised,  and  three  plates  are  made, 
which  are  at  once  placed  in  the  thermostat.  As  early  as  twenty- 
four  hours  later  isolated  gonorrheal  cultures  will  have  developed. 
The  superficial  colonies  present  a  dark,  punctate  center,  from 
which  a  delicate,  finely  granular  deposit  extends  toward  the 
periphery ;  the  deeper  colonies,  of  whitish-gray  color,  possess  a 
nodular  appearance,  and,  in  the  course  of  two  or  three  days, 
assume  the  shape  of  blackberries.  If  inoculations  are  made  from 
the  colonies,  it  will  be  found  that  they  consist  of  a  mucoid, 
viscid  mass. 

Streak- culture  upon  Solidified  Blood-serum  Agar  Slants  (one 
part  of  liquid  human  blood-serum  at  a  temperature  of  40°  C. 
(104°  F. ),  mixed  with  three  parts  of  liquefied  agar-agar  also  at 
a  temperature  of  40°  C.  (104°  F. ),  and  solidified  in  an  oblique 
position). — Luxuriant  growth  takes  place  at  first  in  the  form  of 
individual  gray  colonies  that  later  coalesce  into  a  moist,  glisten- 
ing, viscid-mucous  deposit,  from  the  periphery  of  which  a  thin, 
veil-like  coating  extends.  The  water  of  condensation  is  covered 
by  a  membrane. 

A  good  liquid  culture-medium  is  constituted  by  human  blood- 
serum,  with  the  addition  of  twice  as  much  peptone-bouillon. 
Upon  this  a  superficial  membrane  forms,  while  the  culture- 
medium  itself  remains  almost  entirely  clear. 

In  the  preparation  of  culture-media  animal  blood-serum  may 
be  employed  in  place  of  human  blood-serum,  although  the  gono- 
cocci do  not  thrive  so  well  upon  the  former  as  upon  the  latter. 
If,  also,  gonorrheal  pus  is  smeared  upon  the  surface  of  several 
agar-tubes  covered  with  a  thin  layer  of  human  blood  (blood-agar, 
p.  82),  a  pure  culture  of  gonococci  may  be  obtained.  A  mix- 
ture of  blood-serum  with  urine  has  also  been  employed  with  suc- 
cess in  the  cultivation  of  gonococci ;  further,  a  mixture  of  2 
parts  of  peptone-agar  with  i  part  of  human  acid  urine,  or  of  i 
part  of  ascites-fluid  with  i  part  of  agar  nutrient  medium  of  the 
following  constitution :    Five   per  cent,    peptone,    2   per  cent. 


GONORRHEA.  313 

glycerin,  3^-  per  cent,  agar,  0.5  per  cent,  sodium  chlorid. 
Wassermann  has  recommended  the  following  culture-medium  for 
gonococci :  To  15  cu.  cm.  of  swine  blood-serum,  from  30  to  40 
cu.  cm.  of  water,  from  2  to  3  cu.  cm.  of  glycerin,  and  0.8  gram 
of  nutrose  are  added,  and  all  are  thoroughly  mixed  and  boiled  for 
fifteen  minutes.  It  is  best  to  repeat  the  boiling  on  the  follow- 
ing day.  The  fluid  thus  obtained  is  heated  to  a  temperature  of 
between  50°  C.  (122°  F.)  and  60°  C.  (140°  F.),  and  mixed 
at  the  same  temperature  with  2  per  cent,  peptone-agar  in  equal 
amount. 

Microscopically,  the  gonococci  thus  cultivated  present  exactly 
the  same  appearances  as  those  present  in  gonorrheal  pus.  The 
organisms  survive  in  the  cultures  for  from  four  to  six  weeks. 
They  are  exceedingly  sensitive  to  drying,  disinfectants,  and 
temperatures  above  42°  C.  (107.6°  F.). 

The  specific  pathogenic  significance  of  gonococci  is 

demonstrated  by  the  fact  that  pure  cultures  introduced  into 
the  normal  human  urethra  (of  paralytics)  give  rise  to  true 
gonorrhea.     Animals  do  not  acquire  gonorrhea. 

Bacteriologic  Diagnosis  of  Gonorrhea. — Microscopic 
examination  of  the  urethral  secretion  is  of  the  greatest 
importance,  and  in  doubtful  cases  almost  indispensable. 
With  the  suspected  discharge  (gonorrheal  threads)  dry 
cover-slip  preparations  are  made,  passed  three  times  through 
the  flame,  and  simply  stained  with  an  aqueous  solution  of 
methylene-blue.  The  cocci  and  the  nuclei  of  the  pus-cells 
are  stained  blue,  the  former  more  deeply  than  the  latter. 
The  characteristic  shape  of  the  cocci  (kidney-shaped  or 
biscuit-shaped),  their  position  in  the  leukocytes,  and  their 
failure  to  stain  by  Gram's  method  justify  a  positive  diag- 
nosis of  gonorrhea. 

Dotible  staining  may  be  successfully  effected  if  the  prepa- 
rations are  first  stained  with  an  alcoholic  solution  of  eosin 
(the  eosin  being  absorbed  with  bibulous  paper),  and  then 
subsequently  treated  with  methylene-blue.  Cocci  and  cell- 
nuclei  will  then  appear  blue,  the  cell-bodies  red. 

Cultivation  of  gonococci  for  diagnostic  purposes  is  not 
necessary. 

Although  positive  evidence  of  the  presence  of  gono- 
cocci in  microscopic  preparations  renders  the  diagnosis  of 
gonorrhea  certain,  negative  evidence  from  examination  of 
the  urethral  secretion  must  be  accepted  with  caution.  It 
is  known   that  when  gonorrhea  has  existed  for  a  consider- 


314  CLINICAL  BACTERIOLOGY. 

able  time  gonococci  are  not  always  demonstrable  in  the 
rather  mucoid  secretion  then  present,  but  that,  neverthe- 
less, the  gonorrhea  may  persist  and  still  be  infective.  The 
gonococci  are  situated  in  the  depth  of  the  mucous  mem- 
brane, and  the  superficial  secretion  may  be  entirely  free  from 
them.  It  is,  therefore,  advisable  in  doubtful  cases  to  induce 
irritation  of  the  urethral  mucous  membrane  (through  the 
use  of  beer,  etc.),  and  thereby  to  stimulate  the  secretion. 
Only  when,  after  repeated  examination  and  after  previous 
irritation,  the  secretion  is  found  free  from  gonococci — it 
then  generally  still  contains  other  cocci  in  abundance — 
may  the  gonorrhea  be  considered  as  terminated,  and  only  a 
catarrhal  urethritis  remains. 

Prophylaxis. — Gonorrheal  infection  occurs  almost  ex- 
clusively through  sexual  intercourse  with  those  suffering 
from  the  disease.  It  is  a  noteworthy  fact  that  intercourse 
with  an  individual  suffering  from  gonorrhea  does  not  neces- 
sarily give  rise  to  infection.  It  is,  however,  not  necessary 
to  invoke  a  special  predisposition  to  the  disease  for  those 
who  are  infected,  and,  on  the  other  hand,  a  special  immunity 
for  those  who  escape.  The  gonococcus  must  encounter  a 
lesion  of  the  mucous  membrane  in  order  to  gain  lodgment 
and  to  give  rise  to  gonorrhea.  When  such  a  lesion  is 
wanting,  infection  may  not  take  place,  especially  if  the  gon- 
ococci taken  up  are  soon  removed  mechanically  by  subse- 
quent irrigation  (perhaps  through  the  urine  in  micturition). 
Recovery  from  gonorrhea  predisposes  to  repeated  attack. 
Rigid  sanitary  supervision  of  prostitutes  alone  is  capable 
of  securing  certain  prophylaxis  against  gonorrhea.  As  a 
prophylactic  measure  against  blennorrhea  of  the  new-born, 
instillations  of  astringents  into  the  conjunctivae  are  in  gen- 
eral employ. 

SYPHILIS. 

The  exciting  agent  of  syphilis  is  not  yet  known.  Of 
the  numerous  bacteria  that  have  been  found  and  to  which 
etiologic  significance  has  been  attached,  the  bacilli  described 
by  Lustgarten  seem  worthy  of  mention. 

Lustgarten  in  1884  found  in  syphilitic  lesions  and  discharges 
special  bacilli  that  he  demonstrated  by  means  of  the  following 
method  of  staining :  Sections  of  tissue  hardened  in  alcohol,  or 
cover-slip  preparations  made  from  discharges  and  passed  but  once 


SYPHILIS.  315 

through  the  flame,  are  kept  for  from  twelve  to  twenty-four  hours 
at  room-temperature  and  then  in  a  solution  of  aniline- water  gen- 
tian-violet for  two  hours  more  at  a  temperature  of  40°  C.  (104° 
F. ) .  They  are  next  decolorized  in  absolute  alcohol,  and  are  then 
exposed  for  ten  seconds  to  the  action  oi  ij4  per  cent,  aqueous 
solution  of  potassium  permanganate,  from  which  they  are  trans- 
ferred to  an  aqueous  solution  of  chemically  pure  sulphurous  acid. 
They  are  then  washed  in  water  and  again  immersed  in  the  po- 
tassium-permanganate solution,  but  now  only  for  three  or  four 
seconds,  and  from  this  they  are  transferred  to  the  solution  of 
sulphurous  acid  ;  and  this  is  repeated  until  the  preparation  ap- 
pears entirely  decolorized,  which  usually  is  brought  about  after 
the  manipulations  have  been  repeated  three  or  four  times.     The 


Fig.  69. — Bacillus  of  syphilis  (Lustgarten),  from  a  condyloma;   X  looo  (Itzerott  and 

Niemann). 


preparations  are  then  dehydrated  in  alcohol,  cleared  in  oil  of 
cloves,  and  mounted  in  xylol  Canada  balsam. 

Lustgarten  believed  that  by  this  method  of  decolorization  all 
bacteria  but  the  syphilis-bacilli,  the  tubercle-bacilli,  and  the 
leprosy-bacilli  yield  up  their  stain.  The  last  two,  however,  are 
distinguished  by  their  resistance  to  hydrochloric  acid  and  nitric 
acid,  both  of  which  rapidly  decolorize  the  syphilis-bacilli. 
Lustgarten  found  the  bacilli  stained  in  this  way  in  all  syphilitic 
infiltrates,  and  in  smaller  number  at  the  center  and  in  larger 
number  at  the  periphery,  as  well  as  in  the  adjacent  apparently 
healthy  tissues.  They  rarely  lie  free,  but  singly  or  in  groups  of 
from  two  to  nine  within  large  lymphoid  cells.  On  one  occasion 
Lustgarten  encountered  them  in  the  lumen  of  a  large  lymphatic. 


316  CLINICAL  BACTERIOLOGY. 

On  examination  of  syphilitic  papules  he  found  the  bacilli  between 
the  prickle-cells  of  the  rete  Malpighii.  The  bacilli  are  from  3.5 
to  4. 5  IX  long  and  about  i  /jt  thick,  straight  or  bent,  in  part  irregu- 
larly curved,  and  their  surface  presents  a  wavy  contour.  They 
are  found  constantly  in  the  syphilitic  lesions  in  varying  number, 
but,  on  the  whole,  not  abundantly. 

A  number  of  observers  have  confirmed  Lustgarten's  state- 
ments, while  athers  have  failed  to  find  his  bacilli.  The  signifi- 
cance of  Lustgarten's  observations  was  severely  shaken  when 
Matterstock  and  Alvarez  and  Tavel  found  bacilli  in  smegma 
that  could  be  stained  by  the  method  of  Lustgarten,  and  that 
also  closely  resembled  the  bacilli  of  syphilis  morphologically. 
Doutrelepont  maintained,  subsequently,  that  after  staining  for 
forty-eight  hours  in  aqueous  methyl-violet  solution,  and  decolori- 
zation  with  solution  of  ferric  chlorid  and  alcohol,  the  smegma- 
bacilli  yielded  up  their  color,  whereas  the  Lustgarten  bacilli 
retained  theirs.  Nevertheless,  the  opinion  is  quite  generally 
held  that  the  so-called  bacilli  of  syphilis  are  identical  with  the 
smegma-bacilli,  of  which  not  a  few  take  the  stain  characteristic 
for  tubercle-bacilli.  This  circumstance  must,  further,  be  borne 
in  mind  in  examinations  for  genito-urinary  tuberculosis.  Con- 
fusion between  tubercle-bacilli  and  smegma-bacilli  may  be 
avoided  by  a  knowledge  of  the  fact  that  the  latter  exhibit  less 
resistance  to  the  action  of  hydrochloric  acid  and  nitric  acid  than 
the  former.  The  smegma-bacilli  exhibit  still  less  resistance  to 
the  action  of  alcohol. 

The  bacillus  of  Lustgarten  can  not,  therefore,  be  consid- 
ered as  the  exciting  agent  of  syphilis,  although  it  may 
possibly  bear  some  relation  to  this  disease.  Whether  bac- 
teria are  the  cause  of  syphilis  at  all  is  wholly  doubtful. 
A  bacterial  etiology  for  syphilis  is  generally  assumed  be- 
cause of  the  varied  resemblance  of  its  clinical  manifesta- 
tions to  those  of  other  bacterial  infectious  diseases — as,  for 
instance,  tuberculosis  and  leprosy.  Perhaps,  however,  the 
exciting  agent  of  syphilis  is  of  an  entirely  different  nature. 
It  is  only  certain  that  it  is  an  organized  body,  a  contagium 
vivum,  but  nothing  further  is  at  present  known  with  regard 
to  it.  Investigation  in  this  field  is  attended  with  great 
diflficulties  because  syphilis  is  not  transmissible  to  animals, 
and  experimentation  fails.  According  to  some  observers, 
monkeys  are  said  to  be  capable  of  acquiring  syphilis, 
while  others  deny  this  assertion.  At  least,  it  appears 
that  not  all  varieties  of  monkeys  are  susceptible  to  the 
disease. 


SYPHILIS.  317 

Syphilitic  infection  takes  place  through  the  conveyance 
of  the  syphilitic  virus,  which  is  contained  in  the  degener- 
ated products  of  the  syphilitic  sclerosis,  as  well  as  in  all 
secondary  lesions,  and  in  the  course  of  florid  syphilis  also 
in  the  blood  of  syphilitic  patients.  This  transmissibility  has 
been  demonstrated  repeatedly  by  experiments  on  human 
beings,  and  first  through  the  famous  experiments  of  the 
palatinate  Anonymus.  Inoculations  with  sweat,  saliva, 
urine,  milk,  and  seminal  fluid  of  syphilitics  have  been  un- 
successful. 

The  conveyance  of  the  infecting  material  takes  place  either 
directly,  usually  through  sexual  intercourse,  less  commonly 
through  kissing,  through  the  nursing  of  a  syphilitic  child, 
through  contact  with  syphilitic  lesions  by  the  fingers 
(physicians,  micjwives),  and  the  like  ;  or  indirectly  through 
instruments,  articles,  etc.,  contaminated  by  the  syphilitic 
virus  (house-epidemics  through  eating-utensils  and  drinking- 
utensils,  infection  through  cigar-tubes,  gloves,  etc.).  A 
most  important  role  is  played  by  hereditary  transmission^ 
which  will  be  fully  discussed  later. 

The  portal  of  infection  may  be  constituted  by  any  por- 
tion of  the  skin  or  mucous  membrane  where  a  slight  breach 
of  continuity,  any  lesion  of  the  epithelial  covering,  exists, 
so  that  the  syphilitic  virus  can  penetrate  deeply.  The  virus 
remains  localized  at  the  site  of  infection,  where  it  manifests 
itself  as  the  so-called  prijuary  lesion,  or  hard  chancre.  In 
accordance  with  the  varying  frequency  of  the  different 
modes  of  infection  already  named,  this  lesion  is  most  com- 
mon on  the  genitalia,  but  it  not  rarely  occurs  also  in  other 
situations  :  on  the  lips,  on  the  tonsils,  on  the  nipples,  on  the 
fingers,  etc.  After  a  period  of  incubation  of  not  less  than 
from  three  to  six  weeks,  and  frequently  much  longer,  the 
virus  is  disseminated  throughout  the  entire  body,  and  the 
disease  becomes  generalized.  The  exciting  agents  them- 
selves are  contained  in  the  blood  and  in  the  secondary 
lesions.  A  poison  alone,  absorbed  perhaps  from  the  site 
of  the  initial  lesion,  could  not  give  rise  to  these  secondary 
lesions,  as  the  disease  is  transmissible  indefinitely  through 
them.  The  exciting  agents  further  must  possess  an  extra- 
ordinary length  of  life.  They  must  survive  throughout 
the  whole  duration  of  the  secondary  stage,  extending  over 
many  years,  for  the  disease  is  transmissible  throughout  this 
entire  period.     Only  in  the  tertiary  stage  is  this  transmis- 


318  CLINICAL   BACTERIOLOGY. 

sibility  wanting,  and  the  manifestations  of  this  stage  can 
not  be  dependent  upon  the  virus  in  a  living  form,  or  at  least 
capable  of  multiplication,  and  infectious.  In  the  case  of 
hereditary  syphilis  the  virus  is  taken  up  by  the  blood,  and 
the  primary  lesion,  which  ordinarily  indicates  the  portal  of 
infection,  is  wanting,  and  the  disease  sets  in  at  once  with 
the  secondary  manifestations. 

Immunity. — All  ages  and  all  races  are  equally  suscep- 
tible to  syphilitic  infection.  Natural  immunity  to  the  disease 
in  human  beings  does  not  exist.  Syphilis  is  characterized 
by  a  marked  tendency  to  relapses,  which  often  appear  after 
long  periods  of  latency.  On  the  other  hand,  one  attack  of 
the  disease  confers  protection  against  subsequent  attack — 
that  is,  it  gives  rise  to  immunity.  Renewed  infection  (rein- 
fection) occurs — even  after  disappearance  of  all  previous 
morbid  manifestations — only  exceptionally. 

According  to  a  law  laid  down  by  Colles,  the  mother  who 
gives  birth  to  a  child  syphilitic  through  the  father,  without 
herself  being  attacked,  is  rendered  immune  by  the  fetus. 
By  analogy  with  other  immunizing  procedures,  it  is  easy  to 
understand  that  the  disease-germs  do  not  pass  over  from 
the  child  to  the  mother,  so  that  the  mother  is  not  infected, 
because  the  placental  barrier  is  impassable  to  the  microor- 
ganisms, but  that,  on  the  contrary,  the  toxins  dissolved  in 
the  blood  pass  through  this  barrier  from  the  child  into  the 
maternal  circulation,  and  thus  confer  immunity  upon  the 
mother.  The  mothers  of  syphilitic  children  are  actually 
immune.  They  may  nurse  their  offspring  at  the  breast 
without  being  infected,  whereas  healthy  nurses  nursing  the 
same  children  would  be  infected  with  syphilis.  Such 
mothers  are  thus  certainly  immune.  The  only  point  for 
discussion  is  whether  they  have  been  immunized  by  the 
children.  According  to  the  view  of  a  number  of  syphilog- 
raphers,  they  are  immune  because  they  have  been,  or  are, 
themselves  syphilitic.  Their  condition  of  health  is  only 
apparent.  We  shall  return  to  this  point  later,  in  the  dis- 
cussion of  heredity.  According  to  the  view  just  expounded, 
immunity  to  syphilis  would  always  be  acquired  through 
previous  infection. 

The  specific  therapy  of  syphilis  has  never  been  essen- 
tially cleared  up.  Whether  potassium  iodid  and  mercurials 
operate  by  destruction  of  the  infecting  microorganisms — as 
seems  the  more  probable — or  by  immunizing  infected  indi- 


SYPHILIS.  319 

viduals,  can  not  be  determined  with  certainty,  so  long  as 
the  exciting  agent  of  syphiHs  is  not  known.  The  efforts  to 
check  florid  syphilis  by  means  of  the  serum  of  individuals 
who  have  recovered  from  syphilis,  or  of  animals  into  whom 
syphilitic  products  have  been  injected,  have  each  and  all 
failed  ;  and  for  the  present,  at  least,  they  are  without  any 
experimental  justification. 

Heredity  of  Syphilis. — Children  of  syphilitic  parents 
are  frequently  syphilitic  at  birth,  or  present  sooner  or  later 
evidences  of  hereditary  syphilis. 

The  mode  of  transmitting  the  disease  may,  under  such 
conditions,  be  as  follows  : 

/.  Through  congenital  transmission  from  the  father — that 
is,  transmission  of  the  disease  by  means  of  the  spermato- 
zoids  {paternal  infectiori).  According  to  the  opinion  of 
most  observers,  this  mode  of  transmission  is  actually 
operative,  the  spermatozoid  of  the  syphilitic  father  carrying 
the  disease-germ  into  the  earliest  indication  of  the  fetus. 
This  view,  however,  has  not  been  proved.  Experimental 
attempts  to  induce  syphilis  in  healthy  individuals  by 
inoculation  of  the  seminal  fluid  of  syphilitic  men  have 
been  made,  but  without  inducing  syphilis  in  the  persons 
experimented  on.  The  assumption  of  a  direct  paternal  in- 
fection is  thus  based  solely  upon  the  clinical  experience 
that  syphilitic  men  may  procreate  syphilitic  children  with- 
out infection  of  the  mother.  The  majority  of  clinicians 
accept  this  as  a  fact.  According  to  some,  the  disease  of 
the  fetus  is  always  to  be  ascribed  to  the  father,  and  should 
the  mother  suffer  at  all,  she  is  believed  to  have  first  acquired 
the  infecting  material  from  the  child  infected  by  the  father 
(retroinfection,  choc  en  retour).  It  has  already  been  men- 
tioned that  the  validity  of  this  proposition  has  been  attacked 
from  various  sides.  Distinguished  syphilographers  are  of 
the  opinion  that  no  child  is  syphilitic  in  the  absence  of 
syphilis  in  the  mother  (A.  Wolff)  ;  that  healthy  women  who 
give  birth  to  syphilitic  children,  and  are  thus  immune,  are 
only  apparently  healthy,  but  in  reality  are  infected ;  that 
often,  even  though  primary  or  secondary  manifestations  are 
not  observed,  they  later  suffer  from  tertiary  manifestations. 
If  this  be  true — that  is,  if  in  a  given  case  the  mother  of  a 
syphilitic  child  is  herself  syphilitic — then  the  illustration 
can  not  be  employed  in  support  of  paternal  infection  ;  the 
disease  of  the  child  may  then  arise  also  through  the  mother. 


320  CLINICAL  BACTERIOLOGY. 

From  all  that  is  known  the  question  as  to  the  direct  inheri- 
tance of  syphilis  by  the  child  from  the  father,  although  this 
is  theoretically  quite  conceivable,  and  is  accepted  by  the 
large  majority  of  clinicians  as  actually  occurring,  must  yet 
be  considered  an  open  one. 

2.  Congenital  transmission  from  the  mother — -that  is, 
transmission  of  the  disease  with  the  ovum — is  accepted 
upon  all  sides  as  a  possible  and  frequent  mode  of  con- 
veyance. As  a  matter  of  fact,  the  offspring  of  syphilitic 
women,  if  the  disease  has  not  advanced  to  the  tertiary  stage, 
are  almost  without  exception  syphilitic,  independently  of 
whether  the  father  is  syphilitic  or  healthy. 

J.  Intrauterine  Infection. — If  the  mother,  healthy  at  the 
time  of  conception,  is  infected  with  syphilis  during  the 
period  of  gravidity,  the  child  also  becomes  syphilitic.  Only 
when  the  infection  of  the  mother  takes  place  in  the  last  two 
months  of  pregnancy  are  healthy  children  at  times  born. 
The  fetus,  under  these  conditions,  must  acquire  the  disease- 
germ  from  the  mother  through  the  placental  circulation. 
Whether  a  lesion  of  the  placenta  is  necessary  for  this  to 
take  place,  as  is  assumed  for  all  other  forms  of  intrauterine 
infection,  has  not  yet  been  decided.  According  to  some 
observers,  the  offspring  of  a  healthy  woman  may  be  infected 
in  the  uterus  through  sexual  intercourse  with  a  syphilitic 
man,  and  the  mother,  in  turn,  be  infected  by  the  fetus  ;  but 
this  is  as  yet  undemonstrated.  The  reverse  procedure — 
infection  of  the  woman,  who  then  infects  the  fetus — is  the 
probable  one. 

^.  Extrauterine  infection  during  parturition  or  in  the  first 
days  of  life,  if  the  mother  be  suffering  from  recent  syphilis 
and  the  child  has  remained  uninfected  until  the  moment  of 
birth,  is  altogether  conceivable,  and  probably  also  occurs. 
Whether,  however,  it  plays  an  important  part,  it  is  difficult 
to  decide,  as  probably  the  majority  of  such  cases,  in  which 
a  child  develops  syphilitic  general  manifestations  about  six 
weeks  after  birth,  are  designated  as  hereditary-syphilitic. 
Extrauterine  infection  of  the  new-born  can  be  demonstrated 
by  the  presence  of  a  primary  lesion,  which  is  wanting  in 
placental  (intrauterine)  infection. 


HYDROPHOBIA.  321 


HYDROPHOBIA    (LYSSA;  RABIES). 

The  exciting  agent  of  hydrophobia  is  not  yet  known. 
Nevertheless,  the  specific  treatment  of  this  disease  has  been 
successful,  owing  to  the  genius  of  Pasteur. 

The  susceptibility  for  hydrophobia  exists  among  all 
warm-blooded  animals.  Human  beings  are  infected  through 
the  bites,  in  the  first  place,  of  rabid  dogs,  then  of  cats, 
wolves,  foxes,  jackals,  and  other  animals,  and,  in  rare 
cases,  of  human  beings  suffering  from  hydrophobia.  The 
saliva  must  thus  contain  the  virus  of  the  disease,  and  as  a 
matter  of  fact  this  had  already  been  demonstrated  experi- 
mentally at  the  beginning  of  the  last  century  by  inocula- 
tion of  dogs  from  human  beings.  The  parotid  is  the  gland 
most  concerned,  while  the  remaining  salivary  glands, 
though  virulent,  are  not  so  constantly  so  as  the  parotid. 
The  saliva  of  dogs  contains  the  virus  of  rabies  as  early  as 
two  days  before  the  appearance  of  the  first  symptoms  of 
the  disease.  The  lacrimal  glands,  the  adrenal  glands,  the 
pancreas,  and  the  mammaiy  glands  of  rabid  animals  are 
further  infectious  ;  the  milk,  at  times  is  so  ;  the  blood,  never. 
Besides,  the  central  nervous  system  is  virulent — the  brain 
and  the  spinal  cord  and,  in  a  conspicuous  and  constant 
manner,  the  medulla  oblongata. 

Experiments  on  Animals. — The  saliva  is  not  used  for 
inoculation-experiments  because,  in  addition  to  the  virus 
of  rabies,  it  always  contains  a  number  of  pyogenic  micro- 
organisms that  act  as  a  disturbing  factor.  The  medulla 
oblongata  of  animals  or  individuals  that  have  died  of  rabies 
is  used  exclusively  for  inoculation-purposes.  A  wateiy 
emulsion  is  made  from  a  small  portion  of  the  medulla  ob- 
longata, and  a  few  drops  thereof  are  injected  beneath  the 
dura  mater  or  into  the  anterior  chamber  of  the  eye  in 
dogs,  rabbits,  etc.  After  a  period  of  incubation  of  from 
twelve  to  fifteen  days  the  animals  develop,  with  almost 
absolute  certainty,  the  symptoms  of  rabies.  Subcuta- 
neous injection  is  not  quite  so  trustworthy.  In  order 
to  obtain  positive  results  the  injection  must  be  made 
deeply,  and  preferably  into  the  exposed  and  divided 
muscle-bundle.  Direct  injection  of  the  virus  into  a  periph- 
eral nerve  is  likewise  attended  with  success.  Healthy 
mucous  membranes  (as  of  the  nose  and  the  conjunctiva) 
also    absorb    the   virus.       The    possibility    of  intrauterine 


*  322  CLINICAL  BACTERIOLOGY. 

transmission  of  rabies  has  been  established  experimentally 
in  a  small  number  of  instances. 

Infection  takes  place  by  way  of  the  nervous  system.  If 
the  spinal  cord  of  a  dog  is  divided  transversely,  and  the  virus 
of  rabies  is  injected  into  a  nerve  of  the  hind-paw,  only  the 
cord  below  the  point  of  division  proves  virulent  after  the 
death  of  the  animal.  The  reverse  conditions  prevail  after 
inoculation  of  a  fore-paw.  Having  reached  the  central  ner- 
vous system  from  the  periphery  (site  of  inoculation  or  of 
bite)  through  the  intermediation  of  the  nerves,  the  virus  de- 
scends into  the  peripheral  nerves  of  the  opposite  side.  For 
this  reason,  if  the  disease  has  developed  slowly,  the  nerves 
of  the  uninjured  side  are  also  found  poisonous  in  experi- 
ments on  animals. 

The  as  yet  unknown  excitant  of  rabies  appears  to  exert 
its  influence  through  its  metabolic  .  products.  At  least, 
according  to  Italian  observers,  the  filtrate  through  porce- 
lain of  an  emulsion  of  the  spinal  cord  from  animals  suffer- 
ing from  rabies  induces  paralytic  manifestations  in  dogs. 
Rabies  may,  therefore,  as  suggested  by  Romberg,  be  desig- 
nated a  toxoneurosis. 

The  dissemination  of  the  virus  of  rabies  in  the  course  of 
the  nerve -paths  explains  why  in  human  pathology  the  prog- 
nosis of  the  disease  varies  so  widely  in  accordance  with  the 
number,  the  seat,  and  the  depth  of  the  bite -wounds.  Every- 
thing depends  upon  whether  the  virus  gains  entrance  into 
a  nei-ve  or  not.  Deep  wounds  are,  therefore,  much  more 
dangerous  than  superficial  ones  ;  injuries  in  regions  with  an 
abundant  nerve-supply  (as,  for  instance,  the  finger-pulp) 
more  so  than  in  other  parts  of  the  body.  The  greatest  dan- 
ger is  involved  in  wounds  of  the  head  and  the  face.  From 
these  the  virus  of  rabies  quickly  reaches  the  medulla 
oblongata,  the  main  seat  of  the  disease.  The  morbidity 
and  the  mortality  of  rabies  (the  developed  disease  is  in- 
curable) are,  according  to  the  most  reliable  statistics,  about 
1 6  per  cent,  of  those  bitten. 

Incubation. — The  duration  of  the  period  of  incubation 
depends  upon  the  same  factors  that  have  been  mentioned 
as  significant  in  prognosis.  The  period  is  the  shorter  the 
nearer  to  the  head  the  portal  of  infection  is  situated.  The 
usual  duration  of  the  incubation-period  is  from  twenty  to 
sixty  days.  The  trustworthy  minimum  observed  has  been 
fourteen  days  ;  the  maximum,  eighteen  months. 


HYDROPHOBIA.  323 

Resistance  of  the  Virus  of  Rabies  (Medulla  Oblongata 
of  Dogs  Dead  of  Rabies). — The  virus  is  destroyed  by  ex- 
posure for  an  hour  to  a  temperature  of  50°  C.  (122°  F.) ; 
further,  to  5  per  cent.  carboHc  acid  for  fifty  minutes ;  to 
mercuric  chlorid,  i  :  1000  ;  to  acetic  acid ;  to  potas- 
sium permanganate.  The  spinal  cord  of  (Paris)  rabbits 
dead  of  rabies,  kept  in  dry  air  and  protected  from  putre- 
faction, loses  its  toxicity  only  after  fourteen  or  fifteen  days. 
The  smaller  the  animal,  the  thinner  the  spinal  cord,  the 
more  rapidly  does  the  loss  in  virulence  take  place. 

Immunization  and  Vaccination. — Pasteur  showed  that 
the  virus  of  canine  rabies  slowly  diminishes  in  intensity 
when  inoculated  from  dogs  upon  monkeys  in  a  progressive 
series.  This  gradual  loss  of  virulence  is  distinctly  appreci- 
able in  the  increase  in  the  period  of  incubation.  If  the 
infecting  material  is  reconveyed  from  monkeys  to  rabbits, 
an  increase  in  virulence  takes  place,  which  constantly  aug- 
ments on  further  inoculation  into  rabbits.  The  period  of 
incubation  becomes  shorter ;  and,  finally,  after  the  hun- 
dredth passage  through  the  body  of  the  rabbit,  the  period 
is  not  longer  than  seven  days.  It  was  impossible  to  pro- 
duce a  more  active  virus.  The  virus  retained  its  virulence 
now  unchanged,  and  Pasteur,  therefore,  designated  it  virus 
fixe,  Pasteur  in  this  way  prepared  a  series  of  rabies-viruses 
that,  beginning  with  the  spinal  cord  of  monkeys  and 
progressing  to  the  spinal  cord  of  rabbits  that  succumbed  to 
the  virus  fixe,  possessed  a  steadily  increasing  virulence. 
On  inoculating  successively  dogs  subcutaneously  with  these 
spinal  cords,  of  from  the  lowest  to  the  highest  degree  of  viru- 
lence, the  animals  treated  failed  to  develop  rabies,  but  be- 
came immune  even  to  subdural  infection  with  the  virus  fixe 
and  to  the  bites  of  other  dogs  suffering  from  ordinary 
rabies. 

A  year  later,  in  1885,  Pasteur  and  his  collaborators, 
Chamberland  and  Roux,  developed  a  still  more  practical 
method  of  immunization.  Proceeding  from  the  fact  already 
mentioned  that  the  medulla  of  animals  suffering  from  rabies 
is  completely  deprived  of  its  virulence  in  from  fourteen  to 
fifteen  days  by  desiccation,  they  dried  the  spinal  cords  of 
rabbits  that  had  succumbed  to  the  virus  fixe  for  from  one 
to  fourteen  days  with  all  aseptic  precautions  in  high  ster- 
ilized glass  cylinders.  The  spinal  cord  fourteen  days  old 
had  lost  all  its  virulence ;  that  thirteen  days  old  and  that 


324  CLINICAL  BACTERIOLOGY. 

twelve  days  old,  in  part ;  and  the  others,  successively  less 
and  less.  Through  successive  inoculations  with  these 
gradations  of  spinal  cords  Pasteur  established  complete  im- 
munity in  dogs.  Upon  the  basis  of  these  facts  Pasteur 
proceeded  to  the  vaccination  of  human  beings  therapeuti- 
cally against  rabies.  In  view  of  the  long  period  of  incuba- 
tion of  rabies  in  human  beings  the  attempt  was  justified  and 
hopeful ;  for,  if  it  were  possible  to  establish  immunity 
through  vaccination  immediately  after  the  bite  of  the  rabid 
animal  and  before  the  period  of  incubation  had  elapsed, 
then  it  could  be  hoped  that  the  disease  would  not  break  out. 
The  results  have  completely  confirmed  Pasteur's  anticipa- 
tions. At  first,  the  injections  were  so  made  that  on  the  first 
day  the  spinal  cord  of  rabbits  kept  for  fourteen  days,  on  the 
second  day  that  of  rabbits  kept  for  thirteen  days,  and  so  on 
for  ten  days  until  the  cord  of  rabbits  kept  for  five  days  was 
reached,  were  successively  injected  subcutaneously  into  the 
subject  to  be  treated  (methode  simple).  Pasteur,  however, 
soon  recognized  that  this  procedure  was  not  sufficient  for 
the  severe  cases  with  deep  and  numerous  wounds. 

Vaccination  is  at  present  practised  in  the  Pasteur  Insti- 
tute in  the  following  manner  (methode  intensive)  :  A  piece 
of  spinal  cord  about  three  millimeters  long  is  rubbed  up  in 
sterile  bouillon  and  injected  beneath  the  skin  in  the  hypo- 
chondrium,  and  on  the  first  day  in  the  morning  medulla 
fourteen  and  ten  days  old — an  injection  being  made  on 
either  side — in  the  evening  medulla  twelve  and  eleven  days 
old  ;  on  the  second  day  in  the  morning  medulla  ten  and 
nine  days  old,  in  the  evening  medulla  eight  and  seven  days 
old  ;  on  the  third  day  two  injections  of  medulla  six  days 
old,  and  from  now  on  an  injection  every  twenty-four  hours 
of  the  more  toxic  spinal  cords  up  to  that  three  days  old. 
With  the  spinal  cord  three  days  old  a  new  series  is  begun, 
commencing  with  the  medulla  five  days  old.  Upon  this 
succeeds  a  third,  and  possibly  a  fourth,  series  of  like 
character. 

Vaccine  may  be  prepared  also  by  dilution  with  sterile 
water  (Bardach)  instead  of  by  desiccation.  This  fact  indicates 
that  also  in  the  dried  spinal  cord  the  virus  is  not  actually 
attenuated,  but  only  diminished  in  amount.  As  a  matter 
of  fact,  Pasteur,  after  injecting  desiccated  medulla  into  a 
guinea-pig  and  the  animal  dying  after  thirty  days,  observed 
the   medulla   of  this   animal  destroy  a   second  guinea-pig 


SMALLPOX.  325 

in  exactly  seven  days ;  the  virus  thus  remained  a  virus 
fixe. 

The  immunity  to  rabies  appears  to  persist  for  a  long  time 
— in  dogs  for  two  years. 

Results  of  Pasteur's  Procedure. — The  great  utility  of 
the  method  of  vaccination  against  rabies  is  no  longer  seri- 
ously doubted    by  any  one.     From    1886    to    January   i, 

1894,  14,430  persons  in  all  were  treated  in  the  Pasteur  In- 
stitute, of  whom  J 2  died.  In  the  year  189 1,  394  persons 
were  treated,  the  diagnosis  of  rabies  in  the  biting  animals 
being  established  with  all  possible  certainty  ;  and  not  a 
single  patient  developed  the  disease.  In  1892,  128  persons 
were  treated,  with  i  death.  In  1893,  132  bitten  persons 
were  treated,  of  whom  none  died.  In  the  year  1894,  1387 
persons  were  vaccinated,  of  whom  7  died,  and  in  the  year 

1 895,  1 520  were  vaccinated,  with  2  deaths.*  These  statistics 
require  no  comment :  they  speak  for  themselves. 


SMALLPOX  (VARIOLA). 

Smallpox,  like  syphilis  and  rabies,  is  one  of  the  diseases 
whose  exciting  agents  are  yet  unknown. 

The  virus  of  smallpox  resides  in  the  contents  of  the 
variolous  pustules,  in  the  desiccating  scales  of  the  skin,  in 
the  sputum,  and  in  the  nasal  secretion  of  those  suffering 
from  the  disease.  It  is  transported  with  the  linen  and  the 
clothing  of  the  sick.  Also  the  air  in  the  neighborhood  of 
the  sick  must,  from  clinical  experience,  be  considered 
a  source  of  infection.  Under  suitable  conditions  the  con- 
tagium  may  retain  its  vitality  for  an  exceedingly  long  time, 
apparently  for  years. 

The  portal  of  infection  for  the  contagium  of  smallpox 
has  not  yet  been  definitely  determined.  According  to  the 
common  opinion,  the  disease  is  generally  acquired  through 
direct  or  indirect  contact  with  the  sick,  and  the  skin  would 
thus  seem  to  constitute  the  portal  of  infection.  In  other 
cases  the  disease  may  be  attributed  to  simple  inhalation  in 
the  neighborhood  of  smallpox-hospitals,  etc.  Finally, 
articles  of  food  (such  as  milk)  and  insects  are  thought  to 
convey  the  infection,  which,  under  these  conditions,  would 

*The  figures  for  1896  were  1388,  with  4  deaths;  for  1897,  1521  cases, 
with  6  deaths.— A.  A.  E. 


326  CLINICAL  BACTERIOLOGY. 

take  place  through  the  mouth  or  the  lungs.  Naturally, 
however,  under  any  circumstances  other  modes  of  infection 
can  not  be  excluded. 

Predisposition  and  Immunity. — The  susceptibility  to 
smallpox  exists  at  all  ages.  Even  new-born  children 
have  been  observed  to  present  the  disease.  In  the  great 
epidemics  of  smallpox  that  traversed  Europe  before  the 
institution  of  compulsory  vaccination  the  morbidity  varied 
in  different  localities.  The  local  predisposition  was  the 
greater  the  more  impoverished  the  community.  It  thus  ap- 
pears that  not  merely  the  telluric  conditions  themselves,  but 
rather  the  vital  conditions  and  the  mode  of  life  of  the  people 
living  upon  a  given  soil,  constituted  the  cause  for  the  varying 
distribution  of  the  disease.  The  marked  increase  in  small- 
pox that  regularly  takes  place  in  winter  has  been  considered 
evidence  of  a  temporal  predisposition.  This  may,  however, 
be  readily  explained  by  the  changed  mode  of  life  in  winter 
(with  greater  confinement  in  closed  rooms,  the  wearing  of 
heavier  and  a  greater  amount  of  clothing,  the  greater 
difficulty  of  cleanliness,  etc.),  without  the  necessity  for  in- 
voking a  direct  influence  of  the  weather  upon  the  germ  of 
the  disease. 

Recovery  from  an  attack  of  smallpox  confers  immunity 
that  lasts  on  the  average  about  ten  years.  A  second  attack 
of  the  disease  before  the  expiration  of  ten  years  is  most 
exceptional,  but  has  been  frequently  observed  after  a 
longer  period.  Third  attacks  of  smallpox  have  been  re- 
ported nine  times  in  the  literature,  and  Cantani  reports  one 
case  in  which  seven  attacks  occurred. 

Upon  the  ancient  experience  that  immunity  is  acquired 
by  recovery  from  smallpox  is  based  the  procedure  of  vario- 
lation— that  is,  the  inoculation  of  healthy  persons  with  true 
smallpox  for  purposes  of  immunization,  which  was  formerly 
practised  and  entailed  not  a  few  sacrifices. 

Vaccinia  and  Vaccination. — Edward  Jenner,  a  physician 
of  Berkeley,  in  England,  between  1749  and  1823,  con- 
vinced himself,  after  study  and  investigation  pursued  for 
years,  that  the  pock-disease  of  cows  (vaccinia),  conveyed 
to  human  beings,  protected  them  from  infection  with  variola. 
On  May  14,  1796,  Jenner  vaccinated  a  youth  with  cowpox- 
lymph  obtained  from  the  hand  of  a  maid  who  had  infected 
herself  in  milking  a  cow  suffering  from  cowpox  of  the 
udder.     The  vaccinated  subject  was  protected  against  sub- 


SMALLPOX.  327 

sequent  variolation.  After  a  large  series  of  further  suc- 
cessful vaccinations  Jenner,  in  1798,  published  his  famous 
communication,  in  which  he  announced  the  established  fact 
of  the  protective  influence  of  cowpox-lymph  against  small- 
pox. Since  then,  vaccination  against  smallpox  has  been 
gradually  adopted  in  all  civilized  countries  ;  in  Germany  it 
has  been  made  obligatory  through  a  law  passed  in  1874. 
In  those  countries  in  which  the  general  practice  of  vaccina- 
tion against  smallpox  is  regulated  by  law,  variola,  which 
formerly  was  responsible  for  a  large  proportion  of  the  mor- 
tality, has  almost  completely  disappeared.  Devastating 
epidemics  now  occur  only  in  uncivilized  countries.  Vacci- 
nation against  smallpox  is  practised  in  Germany  in  the  first 
and  twelfth  years  ;  it  consists  in  the  cutaneous  introduction 
of  the  contents  of  the  pocks  of  young  calves  in  a  fresh 
state  or  rubbed  up  with  glycerin  (animal  vaccination).  The 
lymph  obtained  from  the  vesicles  of  inoculated  persons  has 
the  same  effect  (humanized  lymph).  Of  late,  however, 
animal  lymph  is  almost  universally  preferred,  as  the  danger 
of  simultaneous  transmission  of  other  disease-products 
(syphilis,  etc.)  can  not  with  certainty  be  excluded  in  the 
use  of  humanized  lymph.  Animal  lymph  is  obtained  in 
Germany  by  systematic  vaccination  of  calves  in  institutes 
under  State  supervision. 

In  accordance  with  existing  knowledge,  it  can  be  defi- 
nitely accepted  that  cowpox  is  identical  with  variola  in 
human  beings,  and  that  vaccinia  is  only  a  form  of  variola 
mitigated  by  passage  through  the  body  of  the  cow.  Fischer, 
of  Karlsruhe,  succeeded  in  grafting  the  virus  of  variola 
on  the  body  of  the  calf  by  collecting  and  mixing  together 
the  liquid  and  the  contents  obtained  by  cureting  from  vario- 
lous pustules  in  human  beings  in  their  various  phases,  from 
development  to  suppuration.  The  mixture  was  then  rubbed 
into  the  largest  possible  surfaces  (crucial  incisions  and 
scarification).  Fischer  was  able  in  this  way  to  develop  in 
the  calf  directly  with  the  virus  of  variola  typical  pustules 
whose  contents,  subsequently  upon  reconveyance  to  human 
beings,  from  the  third  generation,  proved  to  be  vaccine. 
Vaccination  against  smallpox  thus  readily  adapts  itself  to 
current  views  regarding  immunity,  inasmuch  as  it  represents 
a  protection  against  disease  induced  through  preventive 
inoculation  of  an  attenuated  though  similar  virus. 

As  to  the  nature  of  the  virus  of  variola,  numerous 


328  CLINICAL  BACTERIOLOGV. 

investigations  have  been  made  with  variola  and  with  calf- 
lymph.  In  spite  of  all  the  efforts  that  have  been  made  the 
exciting  agent  of  smallpox  has  not  yet  been  discovered.  It 
is  possible  that  it  does  not  at  all  belong  to  the  class  of 
bacteria,  and  in  any  event  its  requirements  of  the  culture- 
media  are  different  than  are  those  of  the  bacteria  whose 
gowth  has  thus  far  been  successful.  On  ordinary  bacteri- 
ologic  examination  staphylococci,  pseudo-diphtheria-bacilli, 
and  rarely  streptococci  are  found  in  animal  lymph.  It  has 
been  maintained  by  Landmann  that  the  evidences  of  irrita- 
tion that  are  sometimes  apparent  in  marked  degree  around 
the  pustules  are  due  to  the  activity  of  these  pyogenic  cocci. 
On  the  other  hand,  the  Commission  for  the  Investigation 
of  the  Vaccine  Question  ( 1 896)  emphasized  that  the  staphy- 
lococci found  were  of  moderate,  and  only  exceptionally  of 
considerable,  pathogenicity  for  animals,  and  that  virulent 
streptococci  could  no  longer  be  found  in  the  lymph  after 
the  lapse  of  eighteen  days.  The  streptococci  found  in 
older  specimens  are  to  be  looked  upon  as  only  harmless 
skin-epiphytes,  such  as  can  not  rarely  be  isolated  from  bac- 
terial mixtures.  Frosch,  the  reporter  of  the  Commission 
mentioned,  reached  the  conclusion  that  no  etiologic  relation 
exists  between  the  bacteria  of  the  lymph  and  the  irritative 
and  inflammatory  manifestations  of  the  inoculation-pustule, 
and  that  in  the  preparation  of  animal  lymph  any  noteworthy 
reduction  in  the  number  of  germs  or  their  complete  exclu- 
sion even  with  the  observance  of  strict  antisepsis  is  not 
attainable  ;  that,  therefore,  it  is  impossible  to  obtain  an 
unirritating  lymph-supply. 

Varicella  (chickenpox)  has  nothing  to  do  with  the  ex- 
citing agent  of  variola  and  of  vaccinia,  and  recovery  from 
the  disease  does  not  protect  against  smallpox. 


ACUTE  EXANTHEMATA. 

The  exciting  agents  of  the  acute  exanthemata  are,  like- 
wise, thus  far  unknown. 

Measles  is  contagious  in  an  extraordinary  degree.  The 
susceptibility  of  human  beings  to  the  disease  is  exceedingly 
great  between  the  second  and  tenth  years  of  life ;  it  is  less 
in  the  first  year  and  in  adults.  Natural  immunity  to  measles 
practically  appears  not  to   occur.     The  contagium  is  con- 


ACUTE   EXANTHEMATA.  329 

tained  in  the  nasal  mucus,  the  conjunctival  secretion,  and 
the  sputum  of  those  suffering  from  measles,  and,  as 
experimental  inoculation  appears  to  have  shown,  also  in 
the  blood.  The  contagium  is  taken  up  through  contact 
with  the  sick,  or — as  is  generally  accepted  clinically — 
through  inhalation  of  the  exciting  agent.  The  persistence 
of  the  virus  of  measles  is  not  so  marked  as  that  of  small- 
pox or  of  scarlet  fever.  In  a  dry  state  the  virus  of  measles 
is  said  to  persist  for  about  six  weeks.  Measles  is  rarely 
conveyed  by  means  of  articles  of  clothing,  linen,  etc.,  and 
not  for  long  distances.  The  infectivity  of  measles  exists  as 
early  as  the  last  days  of  the  period  of  incubation — which  is 
between  eight  and  ten  days — and  in  the  stage  of  eruption. 
After  the  exanthem  has  entirely  appeared,  the  danger  of  in- 
fection rapidly  subsides. 

In  measles  also  a  large  number  of  bacteria  have  been 
found,  but,  above  all,  again  cocci,  which  have  also  been 
demonstrated  in  the  blood.  None  of  these  observations 
is  worthy  of  consideration,  because  they  depend  upon 
accidental  or  secondary  infection  or  upon  contamination. 

The  hnmunity  established  by  recovery  from  an  attack  of 
measles  is  rather  active  and  enduring.  Only  thirty-six 
cases  are  recorded  in  the  literature  in  which  two  attacks 
occurred,  and  only  one  with  three  attacks. 

Scarlet  Fever. — The  virus  of  scarlet  fever  is  much  more 
resistant  than  that  of  measles.  It  adheres  for  months  to 
the  clothing  worn  and  the  rooms  occupied  by  the  sick.  It 
appears,  further,  to  possess  considerable  resistance  to  tem- 
perature-influences. Infection  with  scarlet  fever,  in  accord- 
ance with  clinical  experience,  occurs  principally  through 
direct  contact  with  the  sick  or  with  articles  belonging  to 
them,  but  also  through  breathing  of  the  air  in  rooms  satur- 
ated with  the  virus  of  the  disease.  The  period  of  incubation  is 
of  varying  duration  (from  two  to  twenty -four  days).  Even 
toward  the  end  of  this  period,  according  to  Gerhardt,  scarlet 
fever  is  infective,  and  it  remains  so  until  the  cessation  of 
desquamation  and  for  weeks  longer.  Wherein  the  infective 
material  resides,  whether  in  the  secretions,  in  the  blood,  or 
in  the  cutaneous  scales,  is  not  known.  •  Some  observers 
claim  to  have  transmitted  scarlet  fever  by  inoculation,  while 
others  have  failed  to  induce  the  disease  experimentally  by 
means  of  the  blood,  the  scales,  etc.  There  is  much  in  the 
clinical  picture  of  scarlet  fever,  especially  the  nephritis,  in 


330  CLINICAL  BACTERIOLOGY. 

favor  of  the  view  that  the  disease  is  a  toxic  infection,  and 
that  the  exciting  agent  is  not  itself  disseminated  into  the 
viscera.  The  relations  between  scarlet  fever  and  diphtheria 
are  well  known.  Scarlet  fever  is  sometimes  complicated 
by  true  diphtheria  with  diphtheria-bacilli  in  the  membranes. 
More  frequently,  however,  scarlatinal  angina  is  due  to 
streptococci.  Altogether  scarlet  fever  exhibits  a  tendency 
to  be  complicated  by  streptococci,  and  the  secondary  sup- 
purative processes  that  not  rarely  occur  are  attributable  to 
these  microorganisms. 

The  susceptibility  of  human  beings  to  scarlet  fever  is 
greatest  between  the  third  and  eighth  years,  though  not  so 
great  as  that  of  measles.  After  the  tenth  year  the  disease 
is  less  common,  and  still  less  so  in  the  first  year  of  life. 

Like  measles,  scarlet  fever  is  more  common  in  winter 
than  in  summer.  Certain  local  influences  in  the  distribu- 
tion of  scarlet  fever  can  not  be  ignored.  Although  the  dis- 
ease is,  on  the  whole,  everywhere  endemic  in  uniform  dis- 
tribution, noteworthy  variations  occur.  Thus,  some  cities 
have  remained  free  from  scarlet  fever  for  thirty,  and  even 
for  fifty,  years,  although  in  constant  communication  with 
other  infected  cities. 

The  immunity  after  recovery  from  scarlet  fever  is  active 
and  enduring.  Only  twenty-nine  cases  in  all  of  second 
attacks  and  four  of  third  attacks  are  recorded  in  the  litera- 
ture. 

The  large  number  of  bacteria  found  in  cases  of  scarlet 
fever,  both  micrococci  and  bacilli,  require  no  particular 
mention.  Streptococci  have  been  found  most  frequently, 
and  these,  as  in  the  case  of  diphtheria,  appear  to  constitute 
a  frequent,  perhaps  a  regular,  complication  of  the  virus  of 
scarlet  fever. 


WHOOPING-COUGH   (PERTUSSIS). 

In  its  infectivity  whooping-cough  stands  close  to  mea- 
sles. Our  knowledge  of  the  virus  of  the  former  disease 
is  yet  quite  scanty.  The  contagium  is  contained  in  the 
breath  and,  especially,  in  the  expectoration  of  children  suf- 
fering from  whooping-cough.  With  the  sputum  it  finds 
its  way  upon  the  linen,  and  it  may  also  be  conveyed 
through  healthy  persons.  Probably  infection  takes  place 
always  through  the  respiratory  tract.     Children  between 


ARTICULAR  RHEUMATISM.  331 

one  and  five  years  of  age  are  most  susceptible  to  whooping- 
cough  ;  the  disease  is  less  common  after  the  tenth  year  of 
life,  although  it  occurs  also  in  adults,  and  not  altogether 
rarely  in  the  first  year  of  life.  Whooping-cough  has  been 
observed  repeatedly  in  new-born  children  whose  mothers 
have  suffered  from  the  disease  toward  the  end  of  gravidity. 
The  disease  is  especially  common  in  spring  and  autumn. 
As  a  rule,  it  attacks  human  beings  but  once.  A  large  num- 
ber of  bacteria  have  been  cultivated  from  the  sputum  of 
patients  suffering  from  whooping-cough,  and  animal  para- 
sites also  have  been  found  therein  ;  the  demonstration  of 
any  etiologic  significance  for  any  of  these  is  yet  lacking. 


ARTICULAR  RHEUMATISM. 

Although  the  exciting  agent  of  articular  rheumatism  is 
entirely  unknown,  there  is,  however,  no  doubt  that  this 
disease  is  infectious.  The  febrile  course,  with  the  general 
manifestations,  the  frequent  complicating  inflammations  of 
serous  membranes  and  the  endocardium,  and  the  secondary 
nephritis,  point  forcibly  to  the  infectious  nature  of  poly- 
arthritis. The  disease  is  well  known  to  be  dependent  in  a 
high  degree  upon  meteorologic  influences,  but  the  necessity 
for  certain  influences  increasing  the  predisposition  also  in 
the  etiology  of  other  infectious  diseases  has  already  been 
demonstrated.  Acute  articular  rheumatism  is  in  no  wise 
contagious.  Rheumatic  polyarthritis  is  one  of  those  infec- 
tions after  whose  termination  immunity  lasts  for  only  a 
short  time,  if  it  occur  at  all,  to  be  replaced  soon  by 
heightened  predisposition.  Frequently  the  same  individual 
suffers  from  repeated  attacks  of  this  disease.  It  is  doubtful 
if  all  cases  that  are  grouped  clinically  under  the  designation 
of  acute  articular  rheumatism  are  associated  etiologically. 
The  fact  that  one-quarter  of  the  cases  are  refractory  to  the 
influence  of  the  known  specifics  (salicylic  acid,  antipyrin) 
appears  indicative  of  etiologic  multiplicity.  In  any  event  it 
is  to  be  borne  in  mind  that  the  clinical  picture  of  poly- 
arthritis may  also  be  produced  by  the  exciting  agents  of 
other  diseases  (gonorrhea,  scarlet  fever). 


332  CLINICAL   BACTERIOLOGY. 


RELAPSING  FEVER. 

The  cause  of  relapsing  fever  was,  in  1873,  found  by 
Obermeier,  a  former  assistant  of  Rudolph  Virchow,  and  who 
died  prematurely,  to  be  a  special  spiral  bacterium,  which 
he  designated  the  spirocheta  of  relapsing  fever.  Obermeier' s 
discovery  was  the  more  far-reaching  because  with  it  an 
organism  of  the  class  of  bacteria  was  for  the  first  time 
recognized  as  the  cause  of  a  human  infectious  disease. 

The  spirilla  of  relapsing  fever  (^spirilla  Obermeieri)  are 
delicate,  wavy  threads,  with  numerous  turns  (from  ten  to 
twenty),  varying  in  length  from  16  to  40  }x,  with  distinctly 
pointed  extremities.     In  form  and  size  they  closely  resemble 


Fig.  70. — Bacillus  of  relapsing  fever,  from  human  blood;  X  looo  (Giinther). 

cholera-spirilla,  though  they  are  only  from  one-half  to  one- 
quarter  as  thick  as  these,  and  they  never  appear  in  the  form  of 
a  comma,  or  in  S-shaped  segments  of  a  screw,  but  always  only  in 
the  form  of  a  complete  screw.  The  spiral  thread  can  not  be 
differentiated  into  individual  members.  The  spirilla  possess 
flagella  and  are  actively  motile.  They  glide  in  rapid,  twisting 
movements  from  one  side  to  the  other  across  the  field  of  vision. 
With  regard  to  their  propagation,  nothing  certain  is  known ; 
probably  this  takes  place  by  division.  The  formation  of  spores 
has  not  been  observed. 

Artificial  cultivation  of  the  spirilla  of  relapsing  fever  upon 
nutrient  media  has  thus  far  not  been  successful.  The  spirilla 
must  be  looked  upon  as  strict  parasites  that  flourish  only  within 


RELAPSING  FEVER.  333 

the  animal  body.  In  leeches  that  have  sucked  themselves  full 
of  the  blood  of  patients  suffering  from  relapsing  fever,  and  have 
been  preserved  upon  ice,  the  spirilla  remained  alive  for  ten  days. 
Little  is  known  with  regard  to  their  temperature-relations.  In 
blood  from  patients  suffering  from  relapsing  fever  kept  in  glass 
tubes  at  a  temperature  of  from  i6°  C.  (60.8°  F.)  to  22°  C. 
(71.6°  F.)  the  spirilla  survive  for  as  long  as  fourteen  days  ;  at 
a  temperature  of  37°  C.  (98.6°  F.),  for  about  twenty  hours; 
at  a  temperature  of  from  39.5°  C.  (103.1°  F.)  to  41.7°  C. 
(107.1°  F. ),  only  from  four  to  twelve  hours  ;  and  at  a  tempera- 
ture of  42.5°  C.  (108.5°  F.),  scarcely  three  hours  (Heyden- 
reich).  Conclusions  with  regard  to  the  growing  bacteria  in  the 
living  blood  can  not  be  drawn  from  these  observations,  as  the 
blood  removed  from  the  body  is  no  longer  a  favorable  nutrient 
medium  for  the  spirilla.     Nothing 

definite  is  known  also  with  regard         O©^/^^  ^ 

to  the  requirements  of  the  spirilla  Q  P-w^     C^ 

for  oxygen.  /f\^ 

The    relapsing   spirilla   can    be      q        ^^^^  \  j/  ^ 

readily  stained  by  means  of  watery       ^^^  f  >    ® 

solutions  of  aniline  dyes ;  they  do         ^  ' 

not  take  acid  stains.  For  the 
demonstration  of  relapsing  spirilla 
in  blood-preparations  the  method        riptff^  ffi^  ^ 

of  Giinther  is  useful ;  this  consists        ^"^^U^  "q  © 
in  introducing  the  dried  cover-slip      ^.^    ,,_spi,och^t^   obermeieri 
preparation,  fixed  by  heatmg,  be-  in  the  blood  (v.jaksch). 

fore  staining  with  gentian-violet  or 

fuchsin,  for  ten  seconds  in  five  per  cent,  acetic  acid,  in  order  to 
extract  the  hemoglobin  from  the  blood-corpuscles.  The  relaps- 
ing spirilla  are  not  so  resistant  as  all  other  bacteria  to  the  action 
of  dilute  potassium  hydroxid  or  concentrated  acetic  acid.  In 
their  reactions  they  resemble  the  protoplasmic  rather  than  the 
nuclear  substances.    The  spirilla  do  not  stain  by  Gram's  method. 

Occurrence  of  Spirilla  of  Relapsing  Fever. — The  spirilla 
are  present,  and  in  varying  number,  only  in  the  blood  of 
those  suffering  from  relapsing  fever,  in  which  they  appear  a 
short  time  before  the  onset  of  the  fever,  and  they  undergo 
considerable  multiplication  during  the  continuance  of  the 
fever,  to  disappear,  a  short  time  before  the  critical  deferves- 
cence, until  the  onset  of  the  next  febrile  paroxysm. 
During  the  afebrile  period  the  spirilla  have  been  found  in  the 
blood  in  but  one  case  by  Naunyn.  They  are  not  ordinarily 
found  in  the  secretions  and  excretions  of  patients  suffering 
from    relapsing   fever,  and  with    equal    rarity  outside    the 


334  CLINICAL  BACTERIOLOGY. 

human  body.  They  have  been  found  once  in  the  urine  in 
a  case  of  relapsing  nephritis.  The  relapsing  spirilla  are, 
therefore,  most  rigid  blood-parasites. 

Transmission  of  Relapsing  Fever. — Relapsing  fever 
has  been  repeatedly  transmitted  to  healthy  human  beings, 
and  with  complete  success  to  apes,  by  means  of  blood  con- 
taining spirilla  (Koch,  Carter,  and  others).  The  spirilla 
can  thus  with  certainty  be  considered  as  the  exciting  agents 
of  relapsing  fever.  How  they  induce  the  disease,  and 
whether  the  fever  is  the  result  of  a  poison  generated  by 
them,  can  not  yet  be  decided  positively.  Equally  little  is 
known  with  regard  to  the  manner  in  which  the  disease  is 
transmitted  naturally.  .  The  disease  is  contagious  ;  as,  how- 
ever, the  spirilla  soon  lose  their  vitality  outside  of  the  body — 
in  water,  on  articles  of  food,  in  the  air — the  ordinary  modes 
of  infection,  by  way  of  the  respiratory  or  the  digestive  tract, 
are  scarcely  to  be  considered.  The  disease  can  be  conveyed 
only  with  the  blood,  as  the  spirilla  apparently  do  not  leave 
this  fluid.  Klebs  refers  to  the  possibility  of  conveyance 
through  blood-sucking  cutaneous  parasites,  but  this  has  not 
been  established  with  certainty.  Intrauterine  transmission 
of  the  spirilla  to  the  fetus  has  been  observed. 

The  mode  of  recovery  from  relapsing  fever,  which,  as 
is  known,  thus  terminates  in  the  majority  of  cases,  is  essen- 
tially not  yet  understood.  According  to  Heydenreich,  the 
bacilli  die  as  a  result  of  the  high  temperature  of  the  patient 
in  the  febrile  attack.  His  observations,  however,  as  has  been 
pointed  out,  do  not  furnish  adequate  evidence  in  support  of 
this  view.  According  to  Metschnikoff,  the  spirilla  collect 
in  the  spleen  during  the  precritical  elevation  of  temperature, 
and  are  there  destroyed  by  phagocytosis.  Baumgarten 
does  not  consider  this  a  healing  process,  but  believes  that 
the  spirilla  are  deposited  in  the  spleen,  and  also  in  the  liver 
and  the  bone-marrow,  where  they  undergo  destruction,  in 
part  through  inclosure  in  leukocytes,  and  in  part  indepen- 
dently of  this  process  ;  but  this  takes  place  only  because 
they  have  ceased  proliferating,  because  their  vital  activity 
has  been  impaired,  and  they  are  nearly  or  already  dead. 
The  cause  of  this  attenuation  Baumgarten  believes  to  re- 
side in  spontaneous  exhaustion  of  the  proliferating  power 
of  the  infecting  microbes,  which  are  endowed  with  only  a 
short  duration  of  life  in  consequence  of  immanent  vital 
laws.       The    successive    febrile    cycles    are  attributed   by 


RELAPSING  FEVER.  335 

Baumgarten  to  new  series  of  generations,  which  arise  from 
individual  spirilla  that  have  not  been  destroyed.  Final 
recovery  may  depend  upon  the  development  of  an  insus- 
ceptibility of  the  blood. 


PART   IV, 


L  MYCOSES  (INFECTIONS  WITH  HLAMENTOUS 
AND  BUDDING  FUNGI)  • 

An  incomparably  lesser  role  in  the  etiology  of  dis- 
ease is  played  by  the  filamentous  fungi  (molds)  and  the 
budding  fungi  than  by  bacteria  (fission-fungi).  The  dis- 
eases generated  by  the  former  are  designated  mycoses. 


MORPHOLOGY  AND  BIOLOGY  OF  THE  FILAMENTOUS 
AND  THE  BUDDING  FUNGI. 

The  filamentous  fungi  are  chlorophyl-free,  thread-like 
cells  that  exhibit  progressive  apical  growth,  and  that,  singly 
or  branched,  usually  divided  into  segments  by  internal 
septa,  unite  to  form  a  deposit  and  at  times  a  dense  felt- 
work  of  closely  interlaced  threads  {Jiyphce).  This  so-called 
vegetative  portion  of  the  fungus  is  known  as  thallus  (fungous 
deposit)  or  myceliwn.  Distinct  from  this  is  the  fructifying 
portion,  the  fruit-beareVy  which  arises  from  the  mycelium 
and  bears  the  fruit  {spores  or  conidid).  The  spores  again 
grow  into  threads ;  the  enlargement  takes  place  steadily 
through  progressive  apical  growth  of  the  threads,  which  in 
turn  give  rise  to  new  spores.  The  structure  of  the  fruit- 
bearing  apparatus  is  so  peculiar  in  a  number  of  filamentous 
fungi  that  this  external  feature  has  been  made  the  basis  of 
their  classification.  Of  the  numerous  species  of  filamentous 
fungi,  running  up  into  thousands,  only  the  following  are  of 
pathologic  interest : 

I.  The  Aspergilli  {Bulbous  Molds'). — The  fruit- hyphae  ex- 
hibit no  division  ;  they  swell  into  the  form  of  a  club  at  the 
extremity.  This  bulbous  enlargement  is  densely  occupied 
by  short,  flask -shaped  structures,  arranged  radially,  the  in- 

336 


FILAMENTOUS  AND   BUDDING  FUNGI. 


337 


termediate  fruit -bearers  {sterig7natd),  upon  which  the  con- 
secutive spores  are  seated. 

2.  The  Penicillii  {Bntsh-7nolds). — The  fruit-bearers,  which 
generally  arise  vertically  from  the  mycelium,  are  trans- 
formed, through  manifold  forked  divisions  in  their  upper 
third,  into  dense  tufts  of  brush-shaped,  ramifying,  delicate 
processes  (basidia),  upon  whose  extremities  the  conidia  are 
seated  in  long  rows  in  the  form  of  globules, 

J.  The  Miicorini  {Glohdar  Molds). — The  fruit-bearers, 
which  are  mostly  unsegmented  and  undivided,  arise  verti- 
cally from  the  mycelium  and  present  at  their  extremity  a 
large  spore-mother-cell  (sporangiuin),  which  is  separated 
from  the  fruit-hypha  by  a  septum  markedly  convex  upward 


©o 


Fig.  72. — A,  Aspergillus  glaucus;  B, 
aspergillus  niger ;  C,  ripe  fructiferous 
head  of  aspergillus  niger  throwing  off 
spores. 


Fig-  73' — Fungi  (penicillium  glaucum). 


{columella).     The  sporangium  contains  within  its  interior, 
separated  by  septa,  the  large  cylindric-oval  spores. 

4.  The  streptothrices  form,  to  a  certain  degree,  a  transi- 
tion between  the  filamentous  fungi  and  the  bacteria.  They 
consist  of  long,  cylindric  filaments,  dividing  by  budding,  and 
from  which  finally  a  true  mycelium  is  formed.  In  many  of 
these  air-hyphae  develop,  which  simply  give  off  special  fruit- 
heads,  spores  {segmentatioit).  These  streptothrix-spores 
must  not  be  placed  upon  the  same  plane  as  the  permanent 
forms  of  the  bacteria.  In  older  cultures  the  streptothrix- 
threads  disintegrate  into  degenerative  products  resembling 
bacteria,  like  rods,  cocci,  spirilla  {fragineniation).  If  these 
structures  are  transplanted  upon  fresh  nutrient  material,  a 
22 


338  CLINICAL  BACTERIOLOGY. 

true  filamentous  network  will  at  once  redevelop.  The 
peculiarities  of  the  strep tothrices  will  be  referred  to  in  detail 
in  the  discussion  of  actinomycosis. 

5.  Oidia  {segmented  molds)  are  likewise  simple  in  arrange- 
ment. They  also  possess  no  special  fruit-heads,  the  spores 
being  detached  directly  from  the  fruit-bearers  that  arise 
from  out  of  the  mycelium.  Their  most  frequent  represen- 
tative is  the  oidium  lactis,  the  white  milk-mold,  which 
vegetates  upon  sour  milk.  The  oidia  form  a  transition  to 
the  so-called  budding  fungi. 

The  budding  fungi  or  yeast-fungi  are  chlorophyl-free 
cells  of  roundish  or  oval  shape  that  multiply  by  budding — 
that  is,  a  small,  roundish,  bud-like  projection  grows  from 
the  periphery  of  the  mother-cell  and,  gradually  increasing 
in  size,  assumes  the  shape  of  the  mother- 
cell,  from  which  it  ultimately  becomes 
detached.  The  newly  formed  cell  under- 
goes the  same  process.  If  the  various 
generations  of  cells  remain  attached  to 
one  another,  long  rows  of  yeast-cells 
result — the  so-called  strings  of  buds. 
Fig.  74.— Yeast-fungi.  Under  special  nutritive  conditions — for 
instance,  upon  solid  culture-media  of 
alkaline  reaction  or  deficient  in  sugar — the  budding  fungi 
also  form  true  mycelial  threads.  (See  Thrush,  p.  351.) 
The  best  known  of  the  budding  fungi  are  the  yeasts 
(saccharomyces  cerevisiae)  and  the  mold  of  wine-must 
(mycoderma  vini). 

Filamentous  and  budding  fungi  flourish  at  room-temper- 
ature. For  their  nutrition  they  require  constantly  pre- 
formed organic  substances,  water,  and,  further,  oxygen  as 
a  rule  ;  a  number  of  the  filamentous  fungi  may  develop  also 
in  the  absence  of  oxygen.  Acid  culture-media  are  pre- 
ferred, although  the  filamentous  and  the  budding  fungi 
grow  also  upon  alkaline  media.  These  fungi  are  present 
everywhere  in  nature,  and  are  always  numerous  in  the  air 
and  upon  articles  of  food.  They  are  capable  of  inducing 
fermentation  (especially,  though  not  exclusively,  the  bud- 
ding fungi)  and  decomposition.  Putrefaction,  which  is 
usually  of  bacterial  origin,  inhibits  the  development  of  fila- 
mentous and  budding  fungi  in  general. 

Microscopic  examination  for  filamentous  and  budding 
fungi  is  made  on   the  whole  in  the  same  way  as  that  for 


FILAMENTOUS  AND   BUDDING   FUNGI.  339 

bacteria.  The  filamentous  fungi,  with  the  exception  of  the 
streptothrices,  which  behave  exactly  as  do  bacteria  with 
regard  to  stains,  do  not  stain  well  in  general,  although  they 
can  be  demonstrated  with  the  aid  of  Loffler's  methylene- 
blue.  Generally,  it  is  preferable  to  examine  these  fungi 
unstained.  The  filamentous  fungi  do  not  take  up  water,  so 
that  they  are  generally  mounted  in  glycerin.  It  is  advisa- 
ble to  undertake  the  demonstration  of  teased  preparations 
from  fungous  vegetations  in  50  per  cent,  alcohol  containing 
a  few  drops  of  ammonia,  in  order  to  avoid  the  disturbing 
influence  of  air-bubbles  ;  or  the  teased  preparations  are 
made  in  Unna's  solution  :  gelatin  i,  alcohol  25,  solution  of 
ammonia  25,  glycerin  25,  water  35.  The  following  recom- 
mendation of  Unna  is  useful  :  The  cover-slip  preparations 
are  placed  for  a  minute  in  5  per  cent,  potassium  hydroxid  ; 
then,  after  rinsing  in  water  for  five  minutes,  in  5  per  cent, 
acetic  acid  ;  and  finally  they  are  exposed  to  the  action  of  a 
strong  aniline  stain  (for  instance,  gentian-violet),  possibly 
under  the  influence  of  heat.  Yeast  is  best  stained  with  a 
dilute  aqueous  solution  of  vesuvin,  as  the  other  aniline 
dyes  readily  give  rise  to  overstaining. 

The  cultivation  of  molds  and  budding  fungi  is  carried  out 
in  the  same  way  as  that  of  bacteria.  Isolation  is  effected 
by  means  of  the  plate -procedure,  preferably  with  acid  gela- 
tin or  agar  (the  gelatin  or  the  agar  is  dissolved  in  acid 
fruit-decoctions,  beer-wort,  or  potato- water  (p.  81)  ),  instead 
of  alkaline  bouillon.  For  further  cultivation  bread-pap  is 
well  adapted  (p.  86). 


PATHOGENIC  ACTIVITY  OF  FILAMENTOUS  AND  BUDDING 
FUNGI  FOR  ANIMALS. 

Although  the  majority  of  filamentous  and  budding  fungi 
vegetate  only  upon  dead  organic  material,  a  small  number 
of  varieties  may  flourish  in  the  animal  body  and  thus  give 
rise  to  disease.  A  special  position  is  occupied  by  the 
streptothrices,  the  most  important  representative  of  which, 
the  streptothrix  actinomyces  (p.  354),  will  be  fully  con- 
sidered. The  best  known  among  the  remaining  pathogenic 
filamentous  fungi  are  the  aspergillus  fumigatus  and  fla- 
vesce7is,  and  the  mucor  corymbifer  and  rhizopodiformis.  If 
an  emulsion  of  these  fungi   in  bouillon  is  injected  into  the 


340  CLINICAL  BACTERIOLOGY. 

ear-vein  of  a  rabbit,  the  animal  will  die  after  the  lapse  of 
two  or  three  days  in  consequence  of  a  general  mold-myco- 
sis. In  all  of  the  organs,  most  abundantly  in  the  kidneys 
and  in  the  liver,  small,  whitish-gray  nodules  are  present, 
which,  when  viewed  microscopically,  are  found  to  consist 
of  a  dense  network  of  mycelial  threads.  These  fungous 
vegetations  never,  however,  contain  fruit-bearers  or  co- 
nidia.  Careful  investigation  has  shown  that  germination  of 
the  injected  spores  has  taken  place,  but  that  fruit-formation 
never  takes  place  in  the  animal  organism. 

By  reason  of  its  deficiency  in  free  oxygen  and  its  alkaline 
reaction  the  organism  does  not  constitute  a  suitable  nutrient 
medium  for  most  molds.  The  overwhelming  majority  of 
these  die  when  introduced  into  the  body,  whether  through 
the  breath  or  in  any  other  way.  The  few  varieties  that 
survive  at  all  prove  pathogenic  only  by  germinating,  and 
acting  as  foreign  bodies  inducing  disturbances  mechanically 
through  irritation  and  vascular  occlusion,  etc.  They  do 
not  undergo  fructification  or  actual  multiplication.  Noth- 
ing likewise  is  known  with  regard  to  any  chemic  activity  on 
the  part  of  molds  or  budding  fungi  in  the  body.  In  a 
certain  sense  the  mold-mycoses  are,  accordingly,  not  true 
infectious  diseases,  as  they  are  not  attended  with  multipli- 
cation of  the  exciting  agent,  and  with  intoxication.  Suc- 
cess in  inoculations  with  pathogenic  molds  depends,  there- 
fore, also  upon  the  number  of  spores  injected.  The  number 
of  disease-foci  corresponds  exactly  with  the  number  of 
spores  introduced,  and  the  animals  die  as  a  result  of  the 
extent  of  the  foci  of  disease  alone.  The  pathogenic  molds 
are,  besides,  inherently  pathogenic,  and  likewise  the  non- 
pathogenic molds  are  always  nonpathogenic.  Augmenta- 
tion or  attenuation  of  pathogenicity  can  not  be  effected. 
The  pathogenic  varieties  are  not  equally  pathogenic  for  all 
animals.  Thus,  the  mucor  corymbifer,  which  destroys  rab- 
bits, is  harmless  in  dogs. 

Pathogenic  molds  are  quite  widely  distributed  in  nature. 
It  is  only  necessary,  for  instance,  to  expose  unsteriHzed 
bread-pap  in  a  closed  beaker  for  one  or  two  days  to  a  tem- 
perature of  from  30°  C.  (86°  F.)  to  40°  C.  (104°  R),  in 
order  to  see  a  dark-green  fungous  coating  form,  consisting 
of  aspergillus  fumigatus.  The  culture  is  generally  pure,  as 
at  a  temperature  of  between  30°  C.  (86°  F.)  and  40°  C. 
(104°  F.)  the  aspergillus  overgrows  all  other  molds.     If, 


FILAMENTOUS  AND   BUDDING  FUNGI.  341 

in  addition,  other  molds  be  present,  a  pure  culture  can  be 
readily  obtained  by  injecting  the  fungous  mass  into  an 
animal.  The  body  of  the  animal  at  once  differentiates 
pathogenic  and  nonpathogenic  molds,  inasmuch  as  the 
latter  are  destroyed  with  certainty.  In  spite  of  this  distri- 
bution of  the  pathogenic  molds,  mycoses  are  not  common 
in  the  animal  kingdom.  Spontaneously  they  occur  rela- 
tively seldom.  Only  in  the  lungs  of  birds  are  fatal  asper- 
gillus-mycoses  and  mucor-mycoses  found  at  all  frequently, 
so  that  a  certain  susceptibility  must  be  assumed  for  this 
particular  tissue,  and  which,  otherwise,  is  generally  wanting 
in  the  animal  body. 

Of  pathogenic  yeasts  but  a  small  number  are  thus  far 
known.  Experimentally  in  animals  they  induce  local  sup- 
puration, tumor-like  swellings,  and  at  times  septic  manifesta- 
tions. 


DISEASES  IN  HUMAN  BEINGS  INDUCED  BY  FILA- 
MENTOUS AND  BUDDING  FUNGI. 

DERMATOMYCOSES  (PARASITIC  DISEASES  OF  THE  SKIN). 

Favus. — The  cause  of  favus  was  recognized  by  Schonlein 
in  1839  as  a  filamentous  fungus,  which,  in  his  honor,  has 
been  named  achorion  ScJionleinii,  and  which  was  the  first  of 
all  organized  causative  agents  of  disease  discovered.  The 
disease  is  characterized  by  a  peculiar  crust  or  scutulum.  On 
microscopic  examination  this  is  found  to  consist  of  a  layer 
of  cornified  epithelial  cells,  beneath  which  there  is  a  mass  of 
fungous  elements,  in  the  form  of  concentrically  arranged 
mycelial  threads,  which  give  off  conidia  toward  the  center 
of  the  favus-body.  The  center  consists  of  conidia  alone  ; 
in  addition  bacilli  and  cocci  are  always  found.  If  a  frag- 
ment of  this  scutulum  be  examined  in  water  or  in  glycerin, 
the  mycelia  will  be  recognized  as  numerously  partitioned  or 
segmented  ramifying  filaments  of  varying  thickness.  The 
Conidia  are  of  variable  form  and  size,  roundish,  oval,  angular, 
in  part  provided  with  a  yellowish  nucleus,  in  part  without 
a  nucleus,  but  with  granular  turbid  contents.  This  fungus, 
which  from  its  form  belongs  to  the  oidium-group,  is  the 
cause  of  favus.  It  is  found  in  association  with  both  favus 
of  the  hairy  scalp  and  in  portions  of  the  body  free  from 
hair,   and  with  favus  of  the   nails.     It  has  been  grown  in 


342  CLINICAL  BACTERIOLOGY. 

pure  culture,  and  with  this  favus  has  been  induced  in  animals 
and  in  human  beings.  It  may  be  mentioned  that  Quincke 
succeeded  in  cultivating  three  distinct  favus-fungi.  More 
recent  investigations  have,  however,  rendered  it  quite  cer- 
tain that  in  all  varieties  of  favus  but  one  fungus  is  found, 
which,  however,  is  not  identical  with  the  exciting  agents  of 
the  other  parasitic  skin-diseases.  The  favus-fungus  grows 
upon  all  nutrient  media,  both  at  the  temperature  of  the  body 
and  at  room-temperature  ;  it  grows  best  somewhat  beneath 
the  surface,  while  only  a  small  number  of  air-hyphse  are 
formed.  The  cultures  are  at  first  whitish,  but  later  they  be- 
come yellow,  and  from  the  periphery  radiating  processes 


Fig.  75. — Achorion  Schonleinii :  X  45° ;  showing  simple  mycelium  in  various  stages 
of  development,  and  free  spores  (after  Duhring). 

extend  into  the  depth  of  the  nutrient  medium.  Microscop- 
ically, a  mycelium  of  branched  hyphae  is  visible.  Individual 
hyphae  swell  at  their  free  extremity  into  the  form  of  a  roll, 
while  others  form  lateral  buds — the  so-called  yellow  bodies 
of  Krai — which  burst,  so  that  their  contents  escape  as  a 
free  body.  At  these  points  the  deep  processes  form.  The 
filaments  themselves  finally  disintegrate  into  oval,  cell- 
shaped  structures. 

Cultural  Properties  of  the  AcJiorion. — On  gelatin-plates 
snow-white,  star-shaped  colonies,  with  an  irregularly  thick- 
ened center,  form.    The  culture-medium  is  quickly  liquefied. 

In  gelatin  stab-cultures  a  thick,  wavy,  superficial  deposit 
appears,  which  is  stained  yellow  beneath  the  surface. 


FILAMENTOUS  AND   BUDDING   FUNGI.  343 

On  agar  a   moderate,  whitish,  wavy  coating  forms,  the 
lower  surface  of  which  exhibits  a  sulphur-yellow  color. 
Upon  agar  and  gelatin  the  mycelium  remains  sterile. 


Fig.  76. — Achorion  invading  root-sheaths  and  bulb  of  the  hair  (Kaposi). 

Upon  blood-serum,  at  a  temperature  of  30°  C.  (Z6^  F.), 
formation  of  conidia  takes  place. 

Favus  occurs    in    dogs,   cats,  and  mice,   as   well  as   in 
human  beings.     It  is  generally  transmitted  to  human  be- 


344 


CLINICAL  BACTERIOLOGY. 


ings  from  others,  less  commonly  through  animals.  On 
the  whole,  the  disease  is  but  slightly  contagious.  Whether 
an  especial  predisposition  on  the  part  of  the  skin  is  neces- 
sary for  the  vegetation  of  the  favus-fungus  is  doubtful. 
Youth  appears  especially  predisposed  to  favus. 

Infection  with  favus  probably  occurs  only  when  the 
fungus  is  deposited  upon  a  macerated  area  of  the  epidermis 
or  gains  entrance  into  a  hair-follicle.  The  morbid  process 
takes  place  beneath  the  epidermis.  It  does  not  extend 
deeply,  and  the  favus-fungus  only  exceptionally  •  reaches 
the  subcutaneous  connective  tissue.  In  rare  instances 
favus  has  been  observed  upon  the  mucous   membrane  of 


Fig-  77- — Trichophyton,  X  450,  as  found  in  epidermic  scrapings  of  ringworm,  showing 
mycelium  and  spores  (after  Duhring). 


the  stomach.  Under  these  circumstances  it  is  probable 
that  the  fungus  has  been  swallowed,  as  opportunity  is 
scarcely  ever  afforded  for  embolic  dissemination. 

The  diagnosis  of  favus  is  made  upon  both  clinical  and 
microscopic  data.  Culture  is  not  necessary  therefor.  If 
it  is  desired  to  make  a  culture,  the  scutulum  is  rubbed  up 
in  a  sterile  mortar  with  sterile  silicic  acid,  and  plates  are 
made. 

Herpes  Tonsurans. — The  various  forms  of  herpes  ton- 
surans upon  the  hairy  scalp  and  upon  parts  of  the  body 
free  from  hair  are,  like  parasitic  sycosis,  eczema  margina- 
tum, onychomycosis  trichophytina,  and  a  number  of  other 
affections  of  the  skin,  due  to  the  presence  of  the  trichophy- 


FILAMENTOUS  AND   BUDDING   FUNGI. 


345 


ton  tonsurans  (discovered  in  1845  by  Gruby  and  Malm- 
sten),  which,  partly  alone  and  partly  in  mixed  infection 
with  bacterial  excitants  of  inflammation,  is  the  cause  of  the 
disease.  The  fungus  appears  especially  in  the  form  of 
extended,  slightly  branched  and  straight  mycelia,  whose 
width  is  less  and  whose  segments  are  comparatively 
much  shorter  than  those  of  the  favus-fungus.  The  conidia 
of  the  trichophyton   resemble  those  of  the   achorion,  al- 


Fig.  78. 


-Root  of  hair  in  tinea  barbse,  showing  early  invasion  of  trichophyton  in  root- 
sheaths  and  upper  part  of  bulb. 


though  they  are  somewhat  smaller  and,  above  all,  less 
numerous.  These  fungous  elements  lie  between  the  epi- 
dermic scales,  between  and  in  the  sheaths  of  the  roots  of 
the  hair.  In  the  less  changed  hairs  the  mycelium  pre- 
ponderates ;  when  the  trichophytic  infiltration  is  consider- 
able, the  spores  are  more  abundant. 

The  trichophyton  may  be    conveyed  to  human  beings 
from  animals.     It  occurs  in  dogs,  cats,  cattle,  and  horses. 


346 


CLINICAL  BACTERIOLOGY. 


More  frequently,  however,  transmission  takes  place  from 
one  human  being  to  another.  Of  all  the  dermatomycoses, 
herpes  tonsurans  is  the  most  contagious.  The  predisposi- 
tion of  the  skin  for  diseases  due  to  the  trichophyton  is  in 
general  greater  than  that  for  favus.  Infection  is  facilitated  by 
all  of  the  conditions  favoring  the  vegetation  of  molds,  such 
as  a  moist  season  of  the  year,  living  in  damp  cellars,  and 
the  like.  The  variability  in  the  clinical  picture  of  the  dis- 
eases due  to  the  trichophyton  depends  in  the  first  place 


Fig.  79. — Microsporon  furfur,  fungus  of  pityriasis  versicolor;  X  700  (Kaposi). 


upon  the  varying  reactivity  of  the  skin  and  the  varied  local- 
izations of  the  fungus,  and  in  the  second  place  also  upon 
the  simultaneous  activity  of  various  bacterial  excitants  of 
inflammation.  The  common  cause  of  all  varieties  of  tricho- 
phytosis has,  however,  been  determined  experimentally 
with  certainty.  In  every  instance  the  trichophyton  was 
cultivated,  and  with  a  pure  culture  thereof  all  the  varied 
forms  of  the  disease  were  reproduced. 

Cultural  Pi'operties  of  the  Trichophyton. — Also  in  culture 


FILAMENTOUS  AND   BUDDING   FUNGI.  347 

the  trichophyton  tonsurans  closely  resembles  the  favus- 
fungus.  The  two  are,  however,  not  entirely  alike.  Lique- 
faction of  gelatin  takes  place  much  more  quickly  with 
the  trichophyton,  and  the  deposits  are  much  greater,  than 
with  the  achorion.  At  a  depth  the  trichophyton  also 
develops  a  yellow  color  in  growth. 

For  microscopic  demonstration  of  the  trichophyton  the 
epidermic  scales,  removed  with  a  sharp  curet,  or  the  epilated 
hairs,  are  treated  with  potassium  hydroxid,  preferably  after 
previous  treatment  with  chloroform  and  ether  for  the  re- 
moval of  fat. 

Pityriasis  Versicolor. — The  cause  of  pityriasis  is  the 
microsporon  furfur  (first  described  by  Eichstedt,  in    1846), 


Fig.  80.— Microsporon  minutissimutn  ;  X  1000  (after  Riehl). 

a  fungus  resembling  the  favus-fungus  and  the  trichophyton, 
and,  likewise,  an  oidium.  The  fungous  masses,  which  are 
at  once  recognizable  in  the  horny  lamella,  removed  by 
means  of  the  finger-nail  or  .a  sharp  curet  and  mixed  with 
six  per  cent,  potassium  hydroxid  upon  a  glass  slide,  consist 
of  unusually  large  and  uniform  conidia,  which  form  regularly 
distributed  masses,  each  of  thirty  or  more,  and  of  slightly 
ramified,  short  mycelia,  which  connect  the  conidial  masses 
with  one  another. 

The  contagiousness  of  pityriasis  is  exceedingly  slight. 
The  microsporon  obviously  requires  a  quite  special  predis- 
position on  the  part  of  the  skin.     It  is  often  found  in  tuber- 


348  CLINICAL   BACTERIOLOGY. 

culous  subjects.  Experimental  transmission  of  the  dis- 
ease has  succeeded  in  several  instances,  but  the  culture  of 
the  fungus  has  not. 

Erjrthrasma,  a  circumscribed  erythema  of  the  skin,  is 
caused  by  the  microsporon  mijiutissirnum,  the  small,  round 
spores  of  which  lie  in  the  superficial  horny  cells,  whereas 
the  mycelium,  consisting  of  small,  delicate,  convoluted 
and  branched  filaments,  in  part  interlacing  and  with  short 
segments,  extends  more  deeply. 

Psoriasis  is  no  longer  included  by  dermatologists  among 
the  parasitic  diseases,  its  supposed  excitant,  the  epidermo- 
phyton,  having  been  recognized  as  a  factitious  product 
(Ries). 

PHARYNGOMYCOSES. 

In  the  pharynx  mycoses  are  not  at  all  rarely  observed  in 
certain  situations.  The  fungous  vegetations  appear  as 
small,  porcelain-white  plugs,  mostly  in  the  lacunae  of  the 
tonsils.  They  consist,  however,  as  a  rule,  not  of  true 
molds,  but  more  commonly  of  filaments  of  the  leptothrix  buc- 
calis  (inycosis  pJiaryngis  leptotHricid)^  whose  botanic  position 
has  not  yet  been  clearly  made  out,  but  which  is  ordinarily 
included  among  pleomorphic  bacteria.  Miller  differentiates 
a  leptothrix  buccalis  innominata,  maxima,  and  gigantea. 
The  leptothrix  is  a  constant  inhabitant  of  the  oral  cavity ; 
and  its  fine  threads,  from  0.5  to  0.8  /i  thick,  are  frequently 
found  on  examination  of  the  sputum.  The  straight,  wavy, 
or  spiral  threads  of  the  leptothrix  are  made  up  of  rod-like 
or  spiral  segments.  In  the  filaments  a  base  and  an  apex 
may  often  be  distinguished  ;  at  their  free  extremities  spher- 
ical formations  appear,  which  in  part  have  been  considered 
arthrospores.  The  appearance  of  this  fungus  is  thus  quite 
similar  to  that  of  the  simple  varieties  of  molds.  Accord- 
ing to  some  observers,  these  detached  *' spores"  may,  like 
true  cocci,  divide  in  pairs.  In  that  event  the  fungus  would 
belong  to  the  bacteria,  and  would  be  included  among  the 
pleomorphic  varieties.  The  leptothrix  stains  yellow  with 
solution  of  iodin  and  potassium  iodid. 

Abundant  opportunity  is  afforded  for  the  entrance  of  the 
fungus  into  the  lacunae  of  the  tonsils,  as  the  inhaled  air  and 
articles  of  food  always  contain  fungi.  In  the  secretion  of 
the  oral  cavity  leptothrix  and  filamentous  fungi  are  always 
present  normally.     The  vegetation   of  the   fungus  in  the 


FILAMENTOUS  AND   BUDDING  FUNGI. 


349 


pharynx  does  not  penetrate  deeply  and  does  not  cause  de 
struction.  Frequently,  it  gives  rise  to  no  subjective  symp- 
toms whatever,  and  only  accidentally  comes  under  observa- 
tion. In  other  instances  it  acts  as  a  mechanical  irritant, 
giving  rise  to  the  sensation  of  a  foreign  body,  and  also  to 
signs  of  slight  inflammation.  The  fungi  adhere  quite  firmly  ; 
they  can  scarcely  be  removed  by  brushing  and  the  like,  but 
the  plugs  must  be  pulled  out  individually  or  removed  with 
the  galvanocautery. 

KERATOMYCOSES,    OTOMYCOSES. 

Keratomycoses  have  been  observed  after  injury  of  the 
intact   cornea  with   soiled  articles   (pitchforks,  etc.),  their 


Fig.  8i. — Aspergillus  fumigatus;  X  500  (Frankel  and  PfeifFer). 


cause  always  appearing  to  be  the  aspergillus  fumigatus. 
The  spores  introduced  through  the  agency  of  the  trauma- 
tism develop  into  filaments  whose  unlimited  growth  is 
capable  of  causing  destruction  of  the  cornea.  The  fungous 
mass  is  surrounded  by  a  wall  of  leukocytes.  Fructifica- 
tion does  not  take  place  under  these  circumstances,  nor 
have  dissemination  of  the  germs  and  the  formation  of  met- 
astases been  observed. 

Otomycoses,  or  myringomycoses,  likewise  occur,  and 
are  also  mostly  due  to  the  aspergillus.     The  presence   of 


350  CLINICAL  BACTERIOLOGY. 

inflammatory  processes  in  the  auditory  canal  favors  the 
lodgment  of  the  fungi,  which  not  only  vegetate  in  the 
secretions  accumulated  in  the  external  auditory  canal,  but 
also  penetrate  the  living  tympanic  membrane. 


PNEUMONOMYCOSES. 

Pneumonomycoses  have  been  observed  repeatedly  in 
human  beings,  and  are  due  to  both  the  aspei^gillus  and  the 
mucor.  Mold-vegetations  have  not  rarely  been  found  in 
bronchopneumonic  foci  at  autopsy,  and  also  during  life  ; 
filamentous  fungi  have  been  found  in  the  sputum,  which 
is  then  sometimes  characterized  by  a  putrid  odor.  In 
general  the  occurrence  is,  however,  rare. 

In  view  of  the  frequency  of  occurrence  of  pathogenic 
molds  in  the  air,  and  of  the  abundance  of  opportunity 
offered  for  the  entrance  of  fungi  into  the  lungs,  it  must  be 
assumed  that  the  human  pulmonary  tissue  possesses  an 
especially  slight  predisposition  for  molds.  It  has  been  men- 
tioned, on  the  other  hand,  that  the  lungs  of  birds  form  a 
more  suitable  nutrient  medium  for  the  vegetation  of  fila- 
mentous fungi,  and  that  birds  not  rarely  succumb  to  spon- 
taneous mycoses. 

The  pulmonary  mycosis  in  human  beings  presupposes  in 
general  a  primary  disease,  a  hemorrhagic  infiltration,  a 
necrosis,  etc.,  of  the  pulmonary  tissue.  The  fungi  pro- 
liferate, as  a  rule,  only  in  such  foci  of  disease.  This 
mycosis  also  is  unattended  with  a  tendency  to  extension, 
and  it  is  unassociated  with  evidences  of  general  infection. 
When  the  primary  disease  undergoes  recovery,  the  tissues 
are  quite  capable  of  disposing  of  the  mycotic  vegetations. 
Pneumonomycosis  often  terminates  in  recovery. 


VISCERAL  MYCOSES. 

Mold-vegetations  in  internal  organs  (kidney,  liver,  etc.) 
have  only  exceptionally  come  under  observation.  The 
reason  for  this  is  readily  made  clear  in  experiments  on  ani- 
mals. The  opportunity  for  infection  is  wanting.  The 
fungi  can  reach  the  organs  in  question  only  through  the 
blood,  and  they  are  scarcely  ever  taken  up  into  the  blood- 
stream from  existing  mycoses,  from  the  intestine,  and 
wherever  else  in  the  body  fungi  are  present. 


THRUSH.  351 


THRUSH. 


Thrush  is  a  local"  disease,  preferably  attacking  mucous 
membranes  lined  with  pavement  epithelium,  and  resulting 
from  the  lodgment  and  proliferation  of  the  thnish-ftingus. 
The  lodgment  of  the  fungus  leads  to  the  formation  of 
milky-white  deposits  varying  from  the  size  of  a  millet-seed 
to  that  of  a  lentil.  These  gradually  undergo  enlargement 
toward  the  periphery,  and  in  the  absence  of  therapeutic 
intervention  coalesce  finally  into  large  membranes. 

Microscopic  Examination  of  Thrush-deposits. — Mi- 
croscopic examination  of  the  white  points  that  represent 
the  first  stage  of  the  thrush-eruption  discloses,  in  addition 
to  squamous  epithelium,  molds  and  bacteria  of  various 
kinds,  always  in  large  number,  the  two  phases  of  the 
thrush-fungus  {oidiiun  albicans) :  the  mycelial  threads  and 
the  conidia.  The  former  are  threads  of  double  contour 
and  of  varying  thickness  and  length,  with  transverse  septa 
and  indentations,  from  which  often  lateral  branches  of 
equal  or  less  thickness  pass  off.  The  conidia,  which  grow 
from  the  mycelia,  at  their  extremities  or  in  the  neighbor- 
hood of  the  septa,  are  more  or  less  spherical,  separate 
readily  from  the  mycelia,  and  lie  among  them,  at  times  iso- 
lated, at  times  in  groups. 

Occurrence  of  Thrush. — Thrush  occurs  most  commonly 
in  new-born  children,  principally  about  the  second  week  of 
life.  In  older  children  and  in  adults  thrush  occurs  only 
when  protracted  disease  (typhoid  fever,  tuberculosis,  etc.) 
has  induced  general  enfeeblement  of  the  organism.  In  the 
new-born,  however,  an  eruption  of  thrush  by  no  means 
presupposes  a  particular  impairment  of  the  general  health. 
The  mucous  membrane  of  the  infant  is  obviously  quite  espe- 
cially predisposed  to  the  vegetation  of  the  thrush-fungus. 
In  experimental  transmission  of  thrush  the  disease  will 
develop  upon  the  healthy  mucous  membrane  of  well- 
nourished  children. 

The  preferable  seat  of  thrush  is  the  mucous  membrane 
of  the  mouth,  and  here  the  tongue,  the  inner  surface  of  the 
lips  and  of  the  cheeks,  are  most  commonly  invaded.  Next 
in  frequency  deposits  of  thrush  appear  upon  the  gums,  in 
the  pharynx,  and  also  in  the  upper  portion  of  the  esopha- 
gus, on  the  anterior  and  upper  surface  of  the  epiglottis,  and 
exceptionally  upon  the  true  vocal  bands.      In  all  of  these 


352  CLINICAL  BACTERIOLOGY. 

situations  pavement  epithelium  is  present,  and  it  has  been 
assumed  that  the  thrush-fungus  is  capable  of  proliferating 
only  upon  mucous  membrane  covered  with  such  epithelium. 
The  thrush-fungus  is  not  rarely  encountered  in  the  vagina 
of  pregnant  women,  which  likewise  is  lined  with  pavement 
epithelium.  It  occurs,  however,  also  upon  mucous  mem- 
branes lined  with  other  kinds  of  epithelium — as,  for  instance, 
that  of  the  stomach,  the  posterior  surface  of  the  epiglottis, 
the  deeper  portions  of  the  respiratory  tract,  although  but 
seldom  in  all  of  .these  situations.  The  thrush-fungus  has 
been  demonstrated  repeatedly  in  bronchopneumonic  foci, 
and  in  numerous  instances  upon  the  mammary  glands  and 
mammillary  areolae  of  nursing  women  whose  children  suf- 
fered from  thrush,  and  in  one  instance  in  encephalitic  sup- 
purative foci  in  a  child  suffering  from  thrush. 

Course  of  Thrush. — The  thrush-fungus  penetrates  the 
uninjured  epithelial  surface,  and  develops  beneath  the  up- 
permost layer  of  epithelium,  within  the  deeper  portions  of 
the  mucosa.  Only  rarely  does  it  invade  the  submucosa. 
The  abundant  fungous  vegetation  raises  the  upper  epithelial 
layer,  and  gradually  separates  it  from  the  connection  with 
the  lower  layers  necessary  for  its  nutrition,  so  that  it  under- 
goes necrosis  and  exfoliation.  The  thrush-membrane  is 
then  exposed  to  view.  The  thrush-proliferation  usually  gives 
rise  to  mild  local -irritative  manifestations.  The  mucous 
membrane  generally  appears  somewhat  altered  at  the  site  of 
the  thrush-deposits.  It  is  dark  red,  slightly  swollen,  dry, 
and  distinctly  sensitive  to  touch.  The  signs  of  an  actual 
inflammation  are,  however,  not  discoverable  microscopi- 
cally. It  has  been  assumed  that  the  thrush-fungus  can 
find  lodgment  only  upon  mucous  membranes  the  seat  of 
catarrhal  inflammation.  This,  however,  does  not  appear 
to  be  correct,  but  the  slight  evidences  of  irritation  may 
really  be  the  result  of  the  purely  mechanical  action  of  the 
fungous  vegetation.  The  saliva  of  patients  suffering  from 
thrush  invariably  has  an  acid  reaction.  There  is  no  sug- 
gestion of  chemic  activity  on  the  part  of  the  thrush-fungus, 
of  any  production  of  toxin.  There  is  an  absence  of  fever 
and,  in  fact,  of  all  constitutional  manifestations.  The 
symptoms  are  of  a  purely  local  nature.  The  fungous 
proliferation  may  become  dangerous  only  when,  by  reason 
of  especially  luxurious  development,  it  obstructs  the 
esophagus  or  the  larynx  ;  and,  further,  in  small  children, 


THRUSH.  353 

through  impairment  of  the  nutrition,  the  tenderness  of  the 
mouth  interfering  with  nursing.  In  general,  however,  the 
disease  is  a  thoroughly  benign  one.  The  thrush- vegeta- 
tions have  no  especial  tendency  to  extend,  and  only  excep- 
tionally have  extension  of  the  thrush-fungus  into  the 
interior  of  blood-vessels  and  transmission  through  emboli 
been  observed.  The  vegetations  can  be  readily  removed 
by  rubbing  the  thrush-plaques  with  soft  cloths  dipped  in  a 
solution  of  borax  (sodium  biborate  2.0,  glycerin  4.0,  dis- 
tilled water  34.0). 

Sources  of  Infection  for  Thrush. — The  thrush-fungus 
is  present  in  the  air  of  dwelling-rooms,  in  the  feces  of  in- 
fants, upon  rubber  nipples,  upon  saccharine  and  amylaceous 
articles  of  food,  etc.,  and  it  may  gain  entrance  into  the 
mouth  with  all  of  these.  Frequently,  as  has  already  been 
mentioned,  the  thrush-fungus  is  found  in  vaginal  mucus, 
and  thrush  in  the  infant  has  been  attributed  to  conveyance 
of  the  fungus  from  the  maternal  genital  canal  during  the 
act  of  parturition.  This  is,  however,  certainly  not  the 
ordinary  mode  of  infection,  as  the  period  of  incubation  of 
thrush,  as  determined  by  numerous  inoculation-experiments, 
is  only  four  or  five  days,  and  the  disease  usually  does  not 
appear  in  infants  before  the  second  week. 

Culture  and  Botanic  Position  of  the  Thrush-fungus. 
— In  accordance  with  the  appearance  of  the  thrush-fungus 
in  the  thrush-membrane  described,  the  fungus  bears  a  close 
resemblance  to  the  varieties  of  oidia  that  cause  the  derma- 
tomycoses,  and  it  has,  therefore,  been  given  the  name 
oiditim  albicans.  Grawitz,  then,  demonstrated  subsequently 
by  culture  that  the  thrush-fungus  is  not  a  mold,  but  rather 
a  budding  fungus.  As  numerous  later  investigations  have 
shown,  the  thrush-fungus  grows  upon  culture-media  of 
acid  reaction  and  containing  an  abundance  of  sugar  (prune- 
decoction  agar)  in  filaments  of  actively  budding  yeast-cells. 
If  these  be  transferred  to  ordinary  meat-peptone  agar — thus 
to  an  alkaline  nutrient  medium,  deficient  in  sugar — distinct 
threads  develop  in  addition  to  the  budding  cells.  Retrans- 
ferred  to  an  acid  medium,  the  threads  again  develop  almost 
exclusively  into  yeast-conidia.  The  oidium  albicans  is  thus 
a  budding  fungus  that,  under  certain  nutritive  conditions, 
forms  hyphae.  Other  observers,  however,  maintain  that 
the  thrush-fungus  is  a  mold.  The  organism  does  not 
liquefy  gelatin.     Upon  gelatin-plates  whitish  colonies  form. 


354  CLINICAL   BACTERIOLOGY. 

and  in  stab-cultures  whitish-yellow  granules  that  send 
radiating  processes  into  the  nutritive  medium.  Upon  agar 
a  whitish-yellow,  wrinkled  deposit  forms  ;  upon  potatoes  a 
dense,  white  coating,  and  upon  bread-pap  a  thin,  white  de- 
posit.    The  temperature-optimum  is  37°  C.  (98.6°  F.). 

It  has  been  demonstrated  experimentally  that  the  thrush- 
fungus  is  the  actual  cause  of  thrush.  It  has  been  possible 
to  develop  thrush  upon  healthy  mucous  membranes  both 
by  means  of  the  bands  of  buds,  and  with  the  filamentous 
cells.  Rabbits  die  in  from  twenty-four  to  forty -eight  hours 
after  intravenous  injection  of  emulsions  of  the  thrush- 
fungus,  a  general  mycosis  resulting,  and  networks  of 
thrush-vegetations  being  found  in  the  kidneys,  the  liver,  etc. 
(G.  Klemperer).  This  pathogenic  action  of  the  thrush- 
fungus  is,  however,  not  constant. 

The  diagnosis  of  thrush  is  made  by  microscopic  exami- 
nation of  the  thrush-deposit ;  cultivation  of  the  fungus  is 
not  necessary. 


ACTINOMYCOSIS. 

The  actinomycosis  of  animals  was  recognized  as  a  dis- 
tinct disease  in  1877  by  Bollinger,  and  in  its  lesions  pecu- 
liar vegetable  structures  (actinomyces,  ray-fungus)  were 
invariably  found. 

Actinomycosis  is  a  disease  peculiar  to  cattle,  having  been  but 
seldom  observed  in  other  animals  (swine,  dogs).  The  usual 
seat  of  the  disease  in  cattle  is  the  lower  jaw,  less  commonly  the 
upper  jaw,  or  also  the  adjacent  soft  parts,  especially  the  tongue. 
The  disease  of  the  jaws  leads  to  the  formation  of  tumors  of 
greater  or  less  extent,  which  subsequently  rupture  outward 
through  the  skin,  less  commonly  into  the  mouth,  and  thus  come 
into  view  as  ulcerated  nodules.  On  section  the  tumor  appears 
pale  yellow ;  it  is  in  general  soft,  but  presents,  in  places,  espe- 
cially softened,  yellowish  areas  of  varying  size,  from  which  on 
scraping  with  the  knife  a  substance  resembling  pus  and  numerous 
yellowish  granules  as  large  as  grains  of  sand  are  obtained. 
Examined  microscopically  the  tumor-mass  represents  granulation- 
tissue;  the  yellow  coloration  is  dependent  upon  abundant  fatty 
degeneration  of  the  cellular  elements.  The  essential  feature  and 
the  peculiar  characteristic  of  the  actinomycotic  nature  of  the  new- 
formation  consist  in  the  yellow  granules  present,  the  so-called 
actinomyces-granules,  to  whose  structure  reference  will  be  made 
later.     These  are  fungous  formations,  and  represent  the  cause 


ACTINOMYCOSIS. 


355 


of  the  disease,  as  has  been  established  with  certainty  by  the 
successful  transmission  to  calves  of  actinomycosis  by  means  of 
such  granules  (Ponfick  and  others). 

The  disease  in  animals  presents,  in  the  first  place,  the  charac- 
ters of  an  inflammatory  new-formation,  which  develops  around 
the  fungous  proliferation  as  a  foreign  body;  besides,  there  is 
considerable  destructive  tendency — the  proliferating  fungi  in 
their  growth  displacing  and  destroying  all  opposing  tissues. 

Infection  in  animals  probably  takes  place  principally  through 
the  mouth  in  feeding.  The  ray-fungus  is  said  to  be  present  in 
feed,  in  wheat-grains,  which  play  an  important  part  in  the  pro- 
cess of  infection.  Infection  through  the  superficial  integument, 
through  the  lungs,  etc. ,  is  possible  also,  but  occurs  less  commonly. 
A  case  of  actinomycosis  of  the  udder  has  been  observed  in  a  pig, 
and  another  of  miliary  pulmonary  actinomycosis  in  a  cow. 


Fig.  82. — Actinomyces  (von  Jaksch). 


Actinomycosis  in  Human  Beings. — Isolated  cases  of 
actinomycosis  in  human  beings  had  already  been  observed 
by  B.  V.  Langenbeck  (1845)  and  by  Lebert  (1857),  but  the 
disease  was  first  clearly  recognized  and  accurately  described 
as  an  independent  affection  by  J.  Israel  in  1878.  Actino- 
mycosis in  hunian  beings  differs  from  the  same  disease  in 
cattle  in  its  slighter  tendency  to  tumor-formation,  and  its 
marked  tendency  to  insidious,  widespread  extension,  which 
may  finally  lead  to  involvement  of  all  of  the  viscera.  The 
chronic  inflammation  that  takes  place  around  the  fungous 
deposit  is  identical  with  that  which  occurs  in  animals.  The 
newly  formed  granulation-tissue  undergoes  fatty  degenera- 
tion more  quickly,  however,  with  disintegration  or  suppura- 
tion, while  the  fungous  proliferation  progresses  and  gives 
rise  to  fistulous  formation,  undermining  the  skin,  penetrat- 


356  CLINICAL  BACTERIOLOGY. 

ing  the  muscles,  and  advancing  without  restraint.  Thus, 
the  fungus  extends  from  the  lower  jaw,  along  the  neck  to 
the  pleurae  and  the  lungs,  and  through  the  diaphragm  into 
the  abdominal  cavity.  Even  invasion  of  the  heart  and  the 
brain  has  been  observed.  In  addition  to  the  continuous 
advance  of  the  disease,  which  usually  is  its  dominating 
feature,  the  dissemination  of  the  fungus  may  take  place 
through  emboli,  in  consequence  of  penetration  of  actino- 
myces  into  the  lumen  of  the  vessels. 

The  peculiar  and  the  single  characteristic  feature  of  all 
actinomycotic  lesions  is  also  in  human  beings  the  presence 
of  the  actinomyces-gramiles.  They  vary  from  the  size  of  a 
grain  of  sand  to  that  of  a  mustard-seed,  and  are  coarse,  dense, 
at  times  calcareous,  bodies  of  yellow  color.  Viewed  with 
low  powers  of  the  microscope  they  appear  as  dark,  finely 
granular  globules,  of  roundish  or  irregularly  nodular 
shape.  If  slight  pressure  is  made  upon  the  cover-glass 
beneath  which  the  granules  lie,  they  are  broken  up  into  a 
number  of  smaller  portions.  Among  the  latter  certain 
radiate  structures  are  especially  characteristic,  the  so-called 
actinomyces-druses  (spheres),  from  which  the  fungus  has  ac- 
quired its  name  of  ray-fungus.  These,  however,  can  be 
observed  only  with  higher  magnifications,  and  best  after 
staining  the  preparation.  From  a  dense  center  there 
radiate  in  every  direction  uniform,  glistening,  variously 
ramified  filaments,  which  enlarge  toward  the  periphery  and 
terminate  in  bulbous,  club-shaped  swellings.  There  thus 
result  star-shaped  figures,  which  have  been  compared  with 
closed  crystalline  druses  or  filled  asters.  In  addition  to 
these,  however,  there  are  always  present  simpler  structures 
radially  branched,  consisting  of  only  a  small  number  of 
filaments.  These  appear  microscopically  as  gray,  viscid, 
sometimes  more  consistent,  granules.  They  are  juvenile 
forms,  which  are  found  with  especial  frequency  in  softened 
foci.  The  filaments  at  times  exhibit  divisions,  and  are 
suggestive  of  strings  of  bacilli.  Finally,  there  are  present 
also  collections  of  spherical  bodies  that  have  been  con- 
sidered as  masses  of  cocci. 

Gram's  method,  as  modified  by  Giinther  (p.  107),  is  well 
adapted  for  staining  the  fungus,  as  is  also  exposure  for  half 
an  hour  to  the  action  of  heated  carbol-fuchsin  solution. 

The  portal  of  infection  for  the  ray-fungus  is  also  in 
human  beings  generally  the   commencement  of  the  diges- 


ACTINOMYCOSIS.  357 

tive  tract,  and  most  frequently,  probably,  carious  teeth,  or 
the  wound  left  after  extraction  of  a  tooth.  The  frequent 
occurrence  of  the  disease  in  the  neighborhood  of  the  jaw, 
and  upon  the  neck,  is  suggestive  of  this  site  of  invasion, 
although  any  other  part  of  the  body  may  constitute  the 
portal  of  entry.  Cases  of  actinomycotic  perityphlitis  have 
been  reported,  and  Chiari  has  recorded  one  of  primary 
actinomycotic  disease  of  the  intestinal  mucous  membrane 
in  an  insane  person,  and  the  like.  It  is  certain  that  an 
injury  of  the  skin  or  of  the  affected  mucous  membrane  is 
necessary  for  infection  to  take  place.  The  source  of  the 
infecting  actinomyces-material  is,  however,  only  rarely 
demonstrable.  The  chewing  of  wheat-grains  is  often 
assigned  as  a  cause.  Some  observers  suggest  the  possi- 
bility of  direct  transmission  from  cattle,  although  this  has 
not  yet  been  demonstrated.  In  a  considerable  number  of 
cases  of  actinomycosis  a  history  of  any  industrial  or  acci- 
dental association  with  cattle  is  completely  wanting. 

The  course  of  actinomycosis  is  exceedingly  insidious. 
The  extension  of  the  chronic  inflammatory  process  that 
attends  the  vegetation  of  the  fungus  can  be  restrained  only 
by  complete  removal  of  the  latter.  The  disease  frequently 
ieads  to  death,  from  the  invasion  of  vital  internal  organs 
(kidneys,  lungs,  etc.)  by  the  fungous  vegetation.  Fre- 
quently, secondary  acute  inflammation,  amyloid  degen- 
eration, etc.,  are  superadded.  Secondary  infection  with 
the  excitants  of  suppuration  appears  especially  common. 
The  extensive  phlegmons,  and  especially  the  pyemia,  which 
often  complicate  the  disease,  can  be  attributed  to  this 
source,  although  thorough  investigations  in  this  connection 
are  wanting. 

The  question  has  not  yet  been  studied  whether  the 
actinomyces  is  capable  of  generating  toxic  substances  and 
whether  it  gives  rise  in  the  body  to  intoxication,  or  whether 
its  action  is  purely  mechanical — that  is,  whether  it  impairs 
the  functions  of  organs  by  invading  their  structure.  It 
is  noteworthy  in  this  connection  that  some  severe  cases 
of  actinomycosis  are  entirely  unattended  with  fever. 

Pure  culture  of  the  actinomyces  has  been  attained  by 
M.  Wolff  and  J.  Israel  (1890).  These  investigators  cultivated 
the  ray-fungus  in  the  absence  of  air,  both  upon  agar  and  in 
hens'    eggs.     Upon  the  surface  of  agar  there  form  at  a  tern- 


358  CLINICAL  BACTERIOLOGY. 

perature  of  37°  C.  (98.6°  F. )  numerous  small,  isolated  nodules 
resembling  dewdrops,  the  first  indications  of  which  are  visible 
with  a  lens  as  early  as  after  two  days,  but  which  do  not  appear 
distinctly  before  from  the  third  to  the  fifth  day.  These  project 
above  the  surface  as  spheres,  though  at  times  they  are  not 
entirely  round.  They  grow  quite  slowly,  often  only  reaching 
the  size  of  a  pinhead  after  eight  days,  and  they  usually  do  not 
grow  larger.  At  the  same  time  they  become  opaque.  As  a  rule, 
they  do  not  become  confluent,  remaining  isolated  for  weeks 
and  months.  If,  after  inoculation,  a  veil-like  turbidity  appears 
on  the  surface  of  the  agar,  the  nodules  described  often  become 
differentiated  subsequently.  At  times,  however,  the  nodules 
coalesce  into  a  whitish  coating.  The  actinomyces-granules 
used  for  inoculation  undergo  enlargement  as  a  whole,  if  not 
carefully  rubbed  upon  the  inoculated  surface,  and  form  a  whitish 
area  in  which,  however,  at  times,  also  a  differentiation  into 
nodules  is  discernible.  In  addition  to  these  fine  nodules  there 
are  present  in  small  number  white  nodules,  rather  larger  than 
lentils,  with  a  blunt,  club-shaped,  projecting  center,  and  which 
become  covered  over  toward  the  periphery,  and  frequently  ex- 
hibit roundish  depressions  at  quite  regular  intervals.  These 
nodules  in  roset-form,  whose  periphery,  further,  is  not  always 
regular,  but  at  times  is  only  slightly  excavated  or  cleft,  are  also 
characterized  by  their  growth  into  the  body  of  the  culture- 
medium  ;  they  send  root-like  processes  into  the  agar.  These^ 
large  nodules  are  not  common,  and  are  usually  encountered 
only  in  small  number  in  culture.  As,  in  addition  to  them,  the 
smallest  nodules  first  described  are  often  present  also,  and  as, 
further,  nodules  of  intermediate  size — transition-forms — with 
root-like  processes  appear,  Wolff  and  Israel  consider  the  char- 
acteristic large  nodules  only  as  the  highest  macroscopic  form  of 
development  of  the  inoculated  actinomyces-fungus. 

In  stab-cultures,  in  addition  to  enlargement  of  the  intro- 
duced granules,  there  develops  throughout  the  entire  length  of 
the  puncture  a  delicate,  gray,  veil-like  turbidity,  and  this  likewise 
contains  numerous  small  nodules.  At  times  large,  flat,  white 
plaques  form  at  the  site  of  puncture. 

The  cultures  described  develop  typically  only  in  the  absence 
of  oxygen ;  otherwise  the  growth  is  but  scanty.  A  tempera- 
ture of  37°  C.  (98.6°  F.)  is  always  necessary  for  the  cultivation 
of  the  fungus,  and  no  development  takes  place  at  temperatures 
between  16°  C.  (60.8°  F.)  and  20°  C.  (68°  F.).  For  this 
reason  growth  does  not  take  place  upon  gelatin. 

In  bouillon  growth  is  quite  scanty.  After  from  three  to  five 
days  white  scales  and  dots  appear  ;  the  bouillon  itself,  like  the 
water  of  condensation  in  agar-cultures,  remains  clear. 

Culture  in  Eggs. — The  actinomyces  thrives  well  in  pigeons* 


ACTINOMYCOSIS.  359 

and  hens'  eggs,  and  when  raw,  as  well  as  when  boiled  for  three 
or  four  minutes.  The  shell  of  the  egg  is  carefully  cleansed  by- 
means  of  mercuric  chlorid  and  sterile  water,  an  opening  is  bored 
into  one  end  by  means  of  a  flamed  needle,  and  a  platinum  wire 
mounted  with  actinomyces-material  is  introduced  deeply  into 
the  egg,  in  which  it  is  moved  to  and  fro  several  times.  The 
opening  made  into  the  inoculated  egg  is  closed  with  sealing- 
wax,  and  the  egg,  with  the  perforated  end  upward,  is  placed  in 
the  thermostat.  After  the  lapse  of  from  nine  to  twenty-eight 
days  the  egg  will  be  found  odorless,  without  formation  of  gas, 
and  not  discolored.  There  will  be  seen  small,  opaque,  whitish 
clumps  and  dots  ranging  in  size  up  to  that  of  a  pinhead,  in  the 
raw  egg,  in  both  the  white  and  the  yolk,  and  in  the  boiled  egg 
at  the  junction  between  the  two ;  or  there  may  be  present  in  the 
liquid  white  of  the  unboiled  egg  lines  and  streaks  of  a  turbid 
mass  resembling  nasal  mucus;  or,  finally,  the  line  of  inoculation 
and  the  central  surface  of  the  coagulated  albumin  may  become 
occupied  by  a  smeary,  granular  mass. 

On  microscopic  examination  Wolff  and  Israel  found  the  agar- 
cultures  made  up  of  short  rods,  mostly  straight,  but  often,  also, 
comma-shaped,  or  even  more  markedly  curved.  These  vary  in 
length  and  thickness,  plump  and  thick  rods  lying  side  by  side 
with  short  and  thin  and  long  and  thick  rods.  Often  the  rods 
present  a  globular  or  an  olive-shaped  enlargement  at  one  ex- 
tremity. In  addition  to  the  rods,  there  are  found  a  small  num- 
ber of  filaments,  rarely  rectilinear,  mostly  wavy  and  curved,  or 
also  spiral.  While  these  are  uncommon  upon  agar,  the  occur- 
rence of  beautiful,  long,  filamentous  networks  is  usual  in 
egg-cultures.  The  filaments,  also,  whose  convolution  often 
exhibits  a  radial  arrangement  at  the  periphery,  at  times  present 
a  knob-like  enlargement  at  the  extremity.  They,  as  well  as  the 
short  rods,  together  with  the  terminal  enlargements,  can  be 
stained  both  by  Gram's  method  and  with  fuchsin.  Filaments 
stained  for  an  hour  with  heated  carbolfuchsin  exhibit  at  "times 
segmentation  into  longer  or  shorter  rods,  ranging  in  size  down  to 
that  of  the  shortest  coccus-like  bodies,  arranged  irregularly,  and 
separated  by  unstained  intervals  of  varying  length.  In  addition 
to  the  filamentous  network,  the  rods  are  also  found  in  the  eggs. 

Finally,  there  are  found  in  agar-cultures,  as  well  as  in 
egg-cultures,  also  micrococcus-like  bodies,  at  times  free,  at 
times  in  dense  masses.  These  are  of  varying  size,  in  part 
spherical,  in  part  oval,  in  part  rather  irregular  and  angular ;  they 
stain  deeply  with  gentian-violet  and  also  by  the  method  of 
Gram.  They  correspond  completely  with  the  granules,  in 
which  the  differentiation  of  the  stained  filaments  can  often  be 
made  out,  and  the  rods  also  when  stained  exhibit  similar  granu- 
lation.     Wolff  and  Israel  do  not   consider  these   coccus-like 


360  CLINICAL   BACTERIOLOGY. 

bodies  as  spores,  on  account  of  their  irregular  shape  and  the 
readiness  with  which  they  take  stains,  but  as  disintegrated  fila- 
ments and  rods.  They  do  not,  however,  represent  dead  detritus, 
for  when  inoculated  upon  fresh  nutrient  media,  they  again  de- 
velop into  rods  and  filaments. 

Experimental  Development  of  Actinomycosis  with 
Pure  Cultures. — The  various  cultures  thus  present  the 
same  structures  as  the  actinomyces-masses  in  the  foci  of 
disease  :  groups  of  cocci,  rods,  and  filaments  ;  the  fully  de- 
veloped clubs  alone  are  wanting.  Culture  shows,  further, 
that  the  cocci,  rods,  and  filaments  represent  various  phases 
of  development  of  one  and  the  same  organism.  Each  form 
is  capable  of  further  development  in  as  many  generations 
as  may  be  desired,  and  in  this  process  the  characteristic 
features  always  appear.  The  actinomyces  retains  its  vitality 
in  culture  for  a  long  time — as  long  as  nine  months. 

That  the  fungus  described  is  the  true  ray-fungus,  in 
spite  of  the  absence  of  typical  clubs,  is  demonstrated  by 
the  successful  inoculation  of  animals  with  cultures.  Wolff 
and  Israel  infected  i8  rabbits,  3  guinea-pigs,  and  i  sheep, 
in  part  with  agar-cultures,  in  part  with  egg-cultures,  by 
intraperitoneal  injection,  or  by  injection  of  a  suspension 
of  the  fungus  into  the  liver.  All  of  the  animals,  after  the 
lapse  of  from  four  to  seven  weeks,  exhibited  multiple  tumors 
in  the  abdomen,  which  contained  typical  actinomyces- 
druses.  With  these  tumors  actinomycosis  could  be  again 
inoculated.  Further,  from  their  contents  new  cultures 
could  readily  be  obtained,  presenting  all  of  the  peculiarities 
of  the  original  culture,  including  absence  of  typical  club- 
forms.  The  chain  of  evidence  in  favor  of  the  specific 
etiologic  significance  of  the  actinomyces-fungus  cultivated 
by  Wolff  and  Israel  is  thus  conclusively  established. 

The  anaerobic  actinomyces-fungus  described  in  the  fore- 
going was  obtained  by  Wolff  and  Israel  in  two  cases  of 
actinomycosis  in  human  beings.  According  to  Kruse,  the 
actinomyces  of  Wolff  and  Israel  has  thus  far  not  been  culti- 
vated by  others.  On  the  other  hand,  it  must  be  pointed  out 
that  one  of  the  authors  of  this  work  has  succeeded  in 
obtaining  the  same  cultures  with  precisely  the  same  char- 
acteristics in  five  cases  of  actinomycosis  in  human  beings. 

A  second  species  of  actinomyces  (aerobic  actinomy- 
ces), which  is  quite  distinct  from  the  actinomyces  of  Wolfif 


ACTINOMYCOSIS.  361 

and  Israel,  has  been  cultivated  by  Bostrom,  Affanassieff, 
and  others.  This  second  variety  of  actinomyces  grows 
more  energetically  in  the  presence  than  in  the  absence  of 
air — an  important  differential  diagnostic  point  in  relation 
to  that  already  described.  The  temperature-optimum  is 
37°  C.  (98.6°  F.),  although  the  fungus  will  grow  well  also 
at  ordinary  room-temperature.  Upon  gelatin-plates,  and 
still  better  upon  agar-plates,  there  form  at  first  fine,  radiat- 
ing colonies,  which  develop  with  comparative  activity  to 
opaque  nodules,  whose  periphery  presents  a  delicate  fila- 
mentous network.  In  agar  streak-culture  a  continuous 
deposit  does  not  form,  as  a  rule,  but  a  series  of  more  or 
less  closely  approximated  nodules.  The  latter  not  rarely 
exhibit  a  brick-red  color  upon  blood-serum,  and  become 
covered  by  a  whitish,  down-like  coating  consisting  of  air- 
hyphae.  From  these  there  develop,  through  a  process  of 
segmentation,  a  series  of  roundish  bodies  arranged  like 
chains,  the  so-called  actinomyces-spores.  These  are  de- 
stroyed only  after  exposure- for  five  minutes  to  a  tempera- 
ture of  75°  C.  (167°  F.),  whereas  the  mature  forms  die  at 
as  low  a  temperature  as  60°  C.  (140°  F.).  When  kept  in 
the  thermostat  for  some  time,  a  wrinkled,  coherent  mem- 
brane eventually  develops  upon  blood-serum,  while  the 
nutrient  medium  is  softened.  Upon  potato  the  aerobic 
actinomyces  grows  as  a  brick-red  deposit,  and  likewise  with 
air-hyphae.  In  bouillon  floating  and  in  part  granular  mem- 
branes form  that  sink  to  the  bottom,  while  the  overlying 
fluid  remains  clear.      Milk  is  gradually  peptonized. 

With  regard  to  the  microscopic  appearances,  the  granules 
and  the  druses,  which  constitute  the  characteristic  feature  of 
the  morbid  process  from  which  the  aerobic  actinomyces  is 
obtained,  resemble  in  every  detail  those  already  described, 
so  that  it  is  only  necessary  to  refer  to  the  account  of  these. 
Preparations  made  from  pure  cultures,  however,  display  great 
differences.  Long  filaments  with  rectangular  branches  may 
be  seen  that  have  arisen  through  the  process  of  budding. 
Terminal  enlargements  are  encountered  but  rarely  in  old 
colonies  that  have  developed  in  the  depth  of  the  culture- 
media.  Transmission  of  the  aerobic  ray-fungus  to  animals 
has  not  yet  been  effected  with  certainty — a  third  point  in  con- 
tradistinction from  the  anaerobic  species  of  Wolff  and  Israel. 

With  regard  to  the  botanic  position  of  the  actinomy- 
ces, it  was  formerly  included  among  the  pleomorphic  bac- 


362  CLINICAL  BACTERIOLOGY. 

teria.  Kruse  (Fliigge's  Microorganisms)  includes  it  among 
the  streptothrices.  These  represent,  to  a  certain  degree,  a 
connecting  Hnk  between  molds  and  bacteria.  They  consist 
of  long,  cylindric  filaments,  dividing  by  budding,  and 
eventually  developing  a  true  mycelium.  Individual  species 
produce  fruit-bearers,  which,  after  the  fashion  of  oidia  (see 
p.  338),  constrict  off  spores  directly,  without  especial  fruit- 
heads.  These  spores  must,  however,  not  be  placed  upon 
the  same  plane  as  the  similarly  designated  permanent  forms 
of  the  bacteria,  as  they  succumb  to  exposure  for  five  min- 
utes to  a  temperature  of  70°  C.  (158°  F.),  as  has  been 
mentioned  in  connection  with  the  aerobic  actinomyces.  In 
old  cultures  the  branching  filaments  break  up  into  bodies 
resembling  bacteria,  bacilli,  cocci,  and  even  spirilla.  If, 
however,  these  disintegrated  products  are  transferred  to  new 
culture -media,  true  filamentous  networks  will  again  develop 
from  them.  According  to  our  own  investigations,  we  have 
likewise  reached  the  conclusion  that  the  entire  group  of 
actinomyces  is  to  be  included  among  the  hyphomycetes. 

The  bacteriologic  diagnosis  of  actinomycosis  requires 
only  microscopic  demonstration  of  the  actinomyces-druses 
in  the  pus.  Culture  is  not  necessary  to  establish  the 
diagnosis. 

Treatment. — According  to  recent  statements,  actinomy- 
cosis is  specifically  influenced  by  potassium  iodid.  It  is 
said  that  recovery  from  the  disease  will  take  place  without 
any  surgical  intervention  upon  administration  of  moderate 
doses  of  this  drug — ^from  two  to  three  grams  daily. 


PATHOGENIC  STREPTOTHRICES. 

In  connection  with  the  actinomyces,  brief  mention  will  be 
made  of  the  streptothrix  Eppinger  and  the  streptothrix  farcinica. 
The  former  was  found  by  Eppinger  in  an  abscess  of  the  brain. 
The  fungus  presented  a  branch  mycelium.  Air-hyphse  and 
spores  are,  however,  found  only  upon  potatoes.  The  fungus 
can  be  stained  by  Gram's  method.  It  grows  best  at  a  tem- 
perature of  37°  C.  (98.6°  F. )  in  the  absence  of  air.  Upon 
gelatin  elevated,  wart-like  yellow  colonies  form,  which  do  not 
liquefy  the  culture-medium.  Upon  glucose-agar  a  wrinkled, 
orange-colored  deposit  forms,  and  upon  potatoes  a  thin,  yellowish- 
red  deposit.  Bouillon  remains  clear,  although  flocculent  islands 
develop.  A  variety  of  pseudo-tuberculosis  develops  in  guinea- 
pigs  and  rabbits  inoculated  with  the  streptothrix  Eppinger. 


PATHOGENIC  YEASTS.  363 

The  streptothrix  farcinica  (also  designated  *'bacille  du  farcin 
des  boeufs  Nocard")  likewise  gives  rise  to  the  formation  of  a 
branched  mycelium,  with  air-hyphae  and  spores.  Growth  takes 
place  between  30°  C.  (86°  F.)  and  40°  C.  (104°  F.)  in  the 
presence  of  air.  The  fungus  can  be  stained  by  Gram's  method. 
Upon  agar  whitish,  dry  scales  form  that  subsequently  become 
yellow  and  finally  confluent.  Bouillon  is  not  rendered  turbid, 
but  presents  flocculi.  A  similar  change  takes  place  in  milk, 
which  is  not  otherwise  altered.  The  farcin  des  boeufs  is  a 
pseudo-tuberculous  affection  of  the  skin  and  the  internal  viscera 
in  cattle.  Experimentally  the  streptothrix  farcinica  induces 
pseudo-tuberculosis  in  cows,  sheep,  and  guinea-pigs. 


PATHOGENIC  YEASTS. 

The  recognition  of  the  pathogenic  yeasts  has  been  made  only 
within  recent  years,  and  is  due  especially  to  the  labors  of  Busse, 
Sanfelice,  Curtis,  and  Rabbinowitsch. 

The  following  varieties  of  pathogenic  yeasts  have  been  found 
in  human  beings: 

The  saccharomyces  hominis  has  been  observed  in  an  infectious 
disease  that  began  with  subperiosteal  inflammation  of  the  tibia, 
and  finally  terminated  in  the  clinical  picture  of  pyemia.  The 
organism  appears  in  the  form  of  round  or  oval  cells,  with  double 
contour  and  a  capsule.  Upon  gelatin -plates  it  forms  round, 
projecting  colonies  that  do  not  cause  liquefaction ;  upon  agar, 
a  whitish  deposit ;  upon  potatoes,  a  grayish-brown  deposit ;  in 
bouillon,  marked  turbidity,  with  the  formation  of  a  membrane; 
and  upon  blood-serum,  a  dewdrop  deposit.  The  saccharomyces 
hominis  possesses  the  property  of  inducing  fermentation  of  grape- 
sugar,  with  the  generation  of  alcohol  and  carbon  dioxid.  It  is 
pathogenic  for  guinea-pigs,  inducing  local  suppuration,  and  for 
mice,  which  die  exhibiting  septic  manifestations. 

The  saccharomyces  subcutaneus  tumefaciens  has  been  cultivated 
from  a  myxomatous  tumor  of  the  thigh.  It  consists  of  oval 
or  round  cells  that  frequently  possess  a  large,  transparent  cap- 
sule. In  gelatin  stab-cultures  development  takes  place  in  the 
form  of  small  colonies ;  the  culture-medium  is  not  liquefied. 
Upon  agar  a  thick,  creamy  deposit  forms,  and  upon  potatoes  a 
whitish  deposit,  later  becoming  brown ;  upon  beerwort-agar  a 
brownish  coating,  and  upon  beerwort,  a  dense  sediment  without 
development  of  membrane-formation.  The  fungus  has  slight  fer- 
mentative activity  for  saccharose,  generating  ethylic  alcohol  and 
acetic  acid.  This  yeast  is  pathogenic  for  white  mice  and  rats,  in 
which  extensive  local  vegetation  occurs.  Microscopically,  the 
tumor-like  formations  present  no  true  structure,  but  are  found 
to  consist  of  enormous  parasitic  infiltrations. 


364  CLINICAL  BACTERIOLOGY. 

n*  INFECTIONS  WITH  THE  LOWEST  FORMS  OF 
ANIMAL  LIFE* 

The  protozoa,  the  lowest  forms  of  animal  life,  have 
hitherto  taken  part  in  the  etiology  of  but  a  small  number 
of  diseases  in  human  beings.  Perhaps  the  future  will  show 
that  they  occupy  a  larger  field  of  activity.  In  a  number 
of  infectious  diseases  whose  exciting  agents  are  as  yet  un- 
known, animal  parasites  are  believed  to  have  been  observed 
— as,  for  instance,  in  whooping-cough,  in  carcinoma,  and 
others.  The  connection  of  disease  with  the  lowest  forms 
of  animal  life  has,  however,  been  established  with  certainty 
only  for  two  affections — namely,  dysentery  and  malaria. 


DYSENTERY    (AMEBIC  ENTERITIS)   AND  TROPICAL  AB- 
SCESS OF  THE  LIVER. 

The  cause  of  dysentery  was  recognized  by  Losch  in 
1 87 1  as  peculiar  animal  parasites  belonging  to  the  class  of 
protozoa — ainebcE — which  were  found  in  dysenteric  stools. 
Koch,  on  the  occasion  of  his  cholera-expedition  to  Egypt, 
noted  the  same  organisms  in  the  base  of  the  ulcers  of  the 
intestine  in  four  fatal  cases  of  dysentery.  Kartulis,  Ogata, 
and  others,  and  in  Germany  recently  Kruse  and  Pasquale, 
and,  further,  Quincke  and  Roos,  have  since  studied  the 
amebae  carefully,  and  confirmed  their  etiologic  relation  to 
dysentery. 

Th  amebaB  of  dysentery  are  unicellular  organisms  of 
varying  size,  with  ameboid  movement.  The  smallest  cells 
are  about  10//  in  diameter ;  the  largest,  50/^  [giant  amebce) ; 
the  majority,  at  rest,  from  20  to  25//.  The  protoplasmic 
body  of  the  amebae  can  be  differentiated  on  movement  into 
an  outer  zone,  the  ectoplasm,  Avhich  is  homogeneous  and 
less  refractive,  and  the  entoplasm,  which  in  part  is  appar- 
ently almost  structureless,  containing  a  small  number  of 
disseminated  granules  and  differentiated  from  the  ectoplasm 
only  by  its  greater  refractive  power,  and  in  part  highly 
granular,  and  completely  filled  with  irregular,  mostly  quite 
fine  granules  (^granular  plasma),  or,  finally,  exhibiting  a 
greater  or  lesser  number  of  large  and  small  vacuoles.  The 
two  layers  are  distinctly  differentiable  from  each  other  only 
when  the  ameba  is  in   motion,  while  the  differentiation  is 


DYSENTERY. 


365 


lost  in  a  state  of  rest.  Frequently,  foreign  bodies,  especi- 
ally red  blood-corpuscles,  also  bacteria,  much  less  com- 
monly leukocytes,  may  be  seen  within  the  entoplasm ;  at 
times  the  protoplasmic  body  is  literally  stuffed  with  blood- 
discs.  The  ameba  invariably  contains  a  nucleus,  which, 
however,  is  not  always  clearly  visible  in  the  moving  cells. 
The  nucleus  is  usually  eccentric,  and  with  change  in  shape, 
often  near  the  periphery.  It  has  a  diameter  of  between  6 
and  8  fi,  is  round  in  shape,  generally  with  a  sharp  contour, 
and  a  punctate  nucleolus.  At  times  the  contents  of  the 
nucleus  are  slightly  granular. 

The  characteristic  of  the  ameba  is  its  mode  of  movement, 
which  is  designated  ameboid.     The  ectoplasm  is  extended  at 


Fig,  83.— Amoebae  coli  in  intestinal  mucus  (after  Losch). 


any  given  point  in  the  form  of  a  blunt,  roundish,  homo- 
geneous process,  and  the  protoplasm  flows  after  it.  In 
this  way  an  actual  change  in  position  can  be  brought  about 
by  a  slow,  at  times  backward,  crawling  movement.  The 
process  may  further  be  retracted,  to  appear  immediately  at 
the  same  or  at  another  point!  The  ameba  is  thus  engaged 
in  constant  change  in  shape.  At. times  the  ectoplasmic 
processes  move  around  the  central  mass  in  waves  without 
locomotion  taking  place. 

Little  is  known  regarding  the  nutrition  of  the  amebae.  In 
general  the  foreign  bodies  so  frequently  seen  in  the  ento- 
plasm, especially  the  red  blood-corpuscles,  are  considered 
as  nutrient  material.     These  are  taken  up  by  a  process  of 


366  CLINICAL  BACTERIOLOGY. 

intussusception.  The  ameba  sends  out  processes — pseudo- 
pods — around  the  foreign  body,  and  in  a  certain  sense  thus 
surrounds  it.  Nothing  is  yet  known  with  certainty  as  to 
the  need  of  oxygen  on  the  part  of  the  amebae. 

Propagation  of  the  amebae  probably  takes  place  by 
dichotomous  division,  although  direct  division  of  cell  and 
nucleus  has  not  yet  been  observed.  Spore -formation,  such 
as  has  been  assumed  by  some,  has  thus  far  not  been  dem- 
onstrated. Of  importance  are  certain  permanent  forms, 
which  have  acquired  especial  resistance  as  a  result  .of  en- 
capsulation ;  these  are  designated  permanent  cysts  or  en- 
cysted amebae.  They  are  generally  small — from  lo  to  \2  fx 
long — have  a  much  sharper,  at  times  distinctly  double, 
contour,  and  possess  a  translucent  luster.  The  nucleus  is 
only  indistinctly  recognizable.  In  form  the  bodies  are 
round,  and  they  are  nonmotile. 

DeatJi  of  the  amebae  is  manifested  principally  by  cessa- 
tion of  movement,  the  greater  or  lesser  activity  of  w^hich 
also  is  an  expression  of  a  corresponding  degree  of  vitality. 
With  the  advent  of  loss  of  motility  the  ameba  invariably 
assumes  a  round  shape,  the  differentiation  between  ecto- 
plasm and  entoplasm  disappears,  and  the  nucleus  becomes 
distinctly  visible.  The  dead  amebae  after  a  time  undergo 
degeneration  :  either  they  become  homogeneous,  with  a 
fat-like  luster,  or  they  disintegrate  into  granules,  often 
after  having  become  constricted  into  several  round  masses. 
In  cover-slip  preparations  the  amebae,  after  cessation  of 
movement — that  is,  after  death — do  not  remain  visible  for 
more  than  two  days ;  in  the  stools  they  have  already  dis- 
appeared within  twenty -four  hours ;  only  the  encysted 
forms  persist  for  a  longer  time,  being  still  distinctly  recog- 
nizable after  twenty  days  in  cover-slip  preparations,  as  well 
as  in  preserved  stools,  but  no  longer  after  four  weeks 
(Quincke). 

The  resistance  of  the  amebae  to  thermal  and  chemic  in- 
fluences has  been  but  little  investigated.  The  most  suitable 
temperature  is  that  of  the  body.  Outside  of  the  organism 
the  parasites  rapidly  lose  their  motility,  which  at  room-tem- 
perature is  lost  after  the  lapse  of  a  few  hours — exceptionally 
eight  hours,  and  in  one  instance  twenty-four  hours.  Ele- 
vation of  the  temperature  to  that  of  the  body  (examination 
upon  a  warm  stage)  increases  the  motility  and  conduces  to 
the  survival  of  the  amebae  for  a  longer  time.      It  has,  how- 


DYSENTERY.  367 

ever,  thus  far  been  impossible,  even  under  the  most  favor- 
able conditions  of  preservation,  to  keep  them  alive  for  more 
than  twenty-four  hours.  Efforts  at  cultivation  of  the 
amebae  upon  any  nutrient  medium  have,  likewise,  thus  far 
been  unsuccessful.  Even  when  they  were  inoculated  in 
pure  culture  no  growth  developed.  (See  Liver-abscess, 
p.  370.)  Kartulis  has  reported  successful  cultivation  of 
dysentery-amebae  in  infusions  of  hay.  His  observations 
have,  however,  turned  out  to  be  incorrect,  as  the  supposed 
dysentery-amebae  were  hay-amebae,  which  always  develop 
abundantly  in  infusions  of  hay  not  sufficiently  sterilized. 

It  may  yet  be  mentioned  that  the  amebae  are  rather  in- 
different to  the  action  of  tannic  acid  (0.3  per  cent.)  and  of 
boric  acid  (i  per  cent),  while  quinin,  in  a  solution  of  i  : 
5000,  speedily  causes  their  death. 

With  regard  to  staining  the  amebae,  reference  may  be  made 
to  the  sections  dealing  with  the  intestinal  changes  below 
and  with  examination  of  the  stools  for  amebae  (p.  371). 

Occurrence  of  Amebae  in  the  Stools  of  Dysenteric 
Patients. — The  amebae  described  are  regularly  present  in 
the  stools  in  cases  of  tropical  dysentery,  and  especially  in 
the  mucous  elements  thereof.  They  vary  in  number.  In 
the  albuminoid  masses  of  mucus,  often  streaked  with  blood, 
found  in  recent  cases,  they  are  present  actually  in  hordes  ; 
in  older  cases,  sometimes  only  in  small  number.  Medicinal 
treatment  may  reduce  the  number  materially,  and  eventually 
cause  their  temporary  disappearance.  The  amebae  can  be 
demonstrated  only  when  the  fresh  stool  is  examined.  Ac- 
cordingly, a  negative  result  in  a  single  examination  of  the 
stools  can  not  be  accepted  as  evidence  of  the  absence  of  the 
amebae.  Only  continuous  and  repeated  examination  of  all 
the  intestinal  evacuations,  especially  at  the  beginning  of  the 
disease,  will  yield  definite  information  as  to  the  presence  or 
the  absence  of  the  amebae. 

The  Intestinal  Alterations  of  Dysentery. — The  clinical 
symptoms  of  dysentery  are  those  of  hemorrhagic  catarrh  of 
the  large  bowel.  The  pathologic-anatomic  conditions  in 
the  intestine  include  the  presence  of  the  dysenteric  ulcer, 
with  elevated,  wall-like,  undermined  borders.  The  ulcera- 
tive process  responsible  for  these  alterations  originates  pri- 
marily in  the  submucosa,  which  undergoes  a  peculiar 
necrotic  transformation.  If  a  section  of  the  intestinal  wall, 
after  hardening  in  absolute  alcohol,  is  stained  with  Loffler's 


368  CLINICAL  BACTERIOLOGY. 

solution  or  by  Gram's  method,  the  amebse  will  be  found 
regularly  in  the  ulcers.  They  lie  almost  exclusively  in  the 
submucosa,  at  times  more  deeply,  upon  the  serosa.  The 
smaller  forms,  about  lyi  times  or  twice  as  large  as  white 
blood-corpuscles,  are  constantly  found  in  the  intestinal  wall. 
They  appear  roundish,  without  a  distinct  nucleus  ;  their 
bodies  stain  a  deep  blue,  and  vacuoles  are  often  indicated. 
In  addition  to  the  amebae,  bacteria  are  always  found  in  the 
floor  of  the  ulcer. 

In  the  intestinal  wall  also  the  amebae,  as  in  the  stools, 
are  only  demonstrable  when  the  tissues  are  subjected  to 
examination  in  a  fresh  state.  At  autopsies  made  four  and 
a  half  hours  after  death  the  amebae  were  present  in  the  in- 
testinal ulcers  in  enormous  number,  while  at  autopsies  made 
eighteen  hours  after  death  but  few  were  present. 

Pathogenic  Activity  of  the  Amebae  of  Dysentery  for 
Animals. — Dysenteric  affections,  with  the  evacuation  of 
hemorrhagic  stools  containing  amebae,  and  with  character- 
istic ulcerative  changes  in  the  large  intestine,  in  which  also 
amebae  are  present,  have  been  induced  experimentally  in 
large  numbers  of  cats  by  injection  of  dysenteric  stools 
through  the  anus.  Losch  had  induced  dysentery  in  a  dog 
in  the  same  way,  by  the  introduction  of  large  numbers  of 
amebae,  both  by  the  mouth  and  by  the  anus.  In  other 
animals,  however,  infection  has  thus  far  not  been  in- 
duced. It  is  noteworthy  that  Quincke  and  Roos  ob- 
served that  of  four  cats  that  received  by  the  mouth  exclu- 
sively stools  containing  amebae,  with  an  abundance  of  the 
encysted  varieties,  two  were  seized  with  typical  amebic 
enteritis.  The  encysted  varieties  thus  appear  to  be  able  to 
escape  the  action  of  the  hydrochloric  acid  of  the  stomach. 

Occurrence  of  Bacteria  in  Dysentery. — In  amebic 
stools,  in  addition  to  amebae,  bacteria  are  always  present ; 
and  especially  and  in  preponderant  number,  streptococci ; 
not  rarely,  also  the  bacterium  coli  ;  and,  further,  the 
bacillus  pyocyaneus,  staphylococci,  etc.  In  the  intestinal 
wall  also  the  amebae  are  found  constantly  in  association  with 
bacteria,  and  here  bacilli  rather  than  streptococci  are  present. 
The  bacteria  lie  within  and  around  the  amebae.  At  times 
they  penetrate  independently  into  the  tissues,  and  more 
deeply  than  the  amebse.  The  association  of  bacteria  and 
amebae  occurs  with  such  regularity  that  some  connection 
must  exist  between  the  two.      It  is  possible  that  the  condi- 


DYSENTERY.  369 

tion  is  one  of  mixed  infection.  This  is,  however,  not  prob- 
able, as  all  of  the  bacteria  found  were  not  virulent  for  the 
animal  on  infection  by  enema,  although  the  pus  from  a 
liver-abscess  that  contains  only  amebae  and  no  bacteria 
will  induce  dysentery  in  cats.  It  is  more  probable  that 
secondary  infection  with  these  bacteria,  all  of  which  are 
regular  inhabitants  of  the  intestine,  has  taken  place.  What 
modification  of  the  clinical  picture  of  the  disease  and  what 
portions  in  the  anatomic  alterations  are  to  be  attributed  to 
the  secondary  bacterial  infection,  and  how  much  is  due  to 
the  amebic  infection  alone  can  not  for  the  present  be  de- 
cided. 

Portals  of  Infection  for  and  Transmission  of  the 
Amebae. — Dysentery  is,  according  to  clinical  experience, 
only  transmissible  in  a  limited  degree  from  one  person  to 
another,  and  from  one  place  to  another.  It  has  not  yet 
been  determined  through  what  medium  the  amebae  gain 
entrance  into  the  body.  The  results  of  the  experiments  of 
Quincke,  already  alluded  to,  render  introduction  by  the 
mouth  possible.  Accordingly,  infection  from  case  to  case 
could  take  place  directly  through  the  intermediation  of  the 
stools.  In  general,  however,  direct  contagion  is  not  con- 
sidered probable  by  clinicians.  It  is  rather  assumed  that, 
as  a  rule,  the  amebae  are  taken  up  from  surrounding 
nature.  Drinking-water  is  believed  often  to  play  the  role 
of  the  intermediary.  As  has  been  pointed  out,  the  amebae 
do  not  persist  in  their  original  form  in  the  stools,  but  they 
quickly  undergo  degeneration.  Whether  they  persist  in 
some  as  yet  unknown  vegetative  intermediate  stage  in 
nature  or  in  another  host,  or  whether  after  leaving  the  body 
they  develop  permanent  forms,  has  not  yet  been  cleared  up. 
Kruse  and  Pasquale  froze  stools  containing  amebae  for  a 
quarter  of  an  hour  in  a  cold  mixture.  After  thawing,  no 
amebae  could  be  detected,  not  even  encysted  forms.  The 
stools,  however,  proved  pathogenic  for  cats,  hemorrhagic 
enteritis,  with  abundant  proliferation  of  amebae,  taking  place 
after  introduction  into  the  bowel.  With  the  disappearance 
of  the  ordinary  forms  of  amebae  observed  the  parasites  evi- 
dently do  not  wholly  perish. 

It  is  an  open  question  how  the  amebae,  when  introduced 

into  the  intestine,  penetrate  the  mucous  membrane  and  gain 

access    to    the   submucosa,   and   whether  a  lesion   of  the 

mucosa  is  necessary  for  this  to  take  place,  or  whether,  per- 

24 


370  CLINICAL  BACTERIOLOGY. 

haps,  with  the  cooperation  of  the  bacteria,  they  are  capable 
of  passing  through  the  intact  mucous  membrane.  Accord- 
ing to  chnical  observation,  exposure  to  cold  and  disorders 
of  digestion  favor  the  development  of  dysentery.  It  thus 
appears  that  an  especial  local  predisposition  for  amebic  in- 
fection is  necessary  in  precisely  the  same  way  as  for  bac- 
terial infection. 

Abscess  of  the  Liver. — Abscess  of  the  liver,  which 
occurs  so  frequently  in  tropical  regions,  has  long  been 
associated  clinically  with  dysentery.  It  has  mostly  been 
considered  as  a  sequel  of  the  latter  disease.  As  a  matter 
of  fact,  the  pus  and  the  abscess-membrane  in  cases  of 
tropical  abscess  of  the  liver  almost  invariably  contain  the 
same  amebae  that  cause  dysentery.  When  the  amebae  are 
absent  from  the  pus,  there  is  generally  no  relation  to  dysen- 
tery ;  so  that  the  presence  of  amebae  may  be  utilized  as  a 
point  in  the  differential  diagnosis  between  idiopathic  and 
dysenteric  abscess  of  the  liver.  Naturally,  careful  and 
repeated  examination  of  the  contents  of  the  abscess  is 
necessary,  as  not  every  specimen  of  the  pus  will  contain 
the  amebae,  which  are  present  in  the  abscess  in  varying 
number. 

Bacteriologic  examination  of  the  pus  from  hepatic  ab- 
scesses containing  amebae  has  yielded  a  positive  result  in 
the  majority  of  cases,,  streptococci,  staphylococci,  colon- 
bacilli,  and  other  bacteria  being  found  present  also.  Only 
a  small  proportion  of  the  abscesses  are  sterile.  If  the  pus 
free  from  bacteria  contains  living  amebae,  practically  a  pure 
culture  is  present,  which  may  then  be  employed  experi- 
mentally. 

It  is  an  open  question,  further,  what  role  in  the  devel- 
opment of  abscesses  of  the  liver  is  played  by  the  bacteria, 
as  all  of  them  possess  pyogenic  activity.  It  is  possible 
that  the  amebae  injure  the  liver-tissue,  and  that  the  bacteria 
then  secondarily  induce  suppuration.  It  is  a  question,  also, 
as  to  how  the  amebae  gain  entrance  into  the  liver.  In  this 
connection  the  portal  circulation,  the  peritoneum,  the  lym- 
phatic vessels,  and  the  biliary  passages,  must  be  considered 
as  possible  media  of  infection.  Dissemination  of  the 
amebae  through  the  blood-stream  appears  the  more  prob- 
able, as  they  have  been  found  repeatedly  in  the  intestinal 
wall,  within  the  lumen  of  vessels.  Penetration  of  the  in- 
testinal wall  and  infection  from  the  peritoneum  also  appear 


AMEB^   IN   NORMAL  INTESTINAL  CONTENTS.         371 

probable.  Amebae  have  been  encountered  in  peritonitic 
exudates,  and  hepatic  abscesses  are  frequently  quite  super- 
ficial and  preferably  in  the  right  lobe.  Dissemination  through 
the  lymph-channels  appears  improbable  by  reason  of  the 
numerous  lymph-glands  interposed. 

Finally,  some  observers  consider  hepatic  abscesses  as  not 
always  the  sequel  of  dysentery,  but  both  diseases  as  due 
to  a  common  cause,  and  of  which  one  may  at  times  pre- 
cede the  other,  and  vice  versa.  If  primary  amebic  ab- 
scesses of  the  liver  actually  occur,  and  are,  perhaps,  fol- 
lowed subsequently  by  dysentery,  migration  of  the  amebae 
through  the  biliary  passages  would  naturally  have  to  be 
assumed  under  such  conditions. 

Amebae  in  Normal  Intestinal  Contents. — In  the  stools 
of  healthy  persons  there  occur,  usually  in  small  number, 
and  sometimes  in  rather  considerable  number,  amebae 
that  are  indistinguishable  microscopically  from  those  that 
give  rise  to  dysentery.  Thus,  amoeba  coli  (Kruse  and 
Pasquale)  or  amoeba  intestini  vulgaris  (Quincke  and  Roos) 
is  not  pathogenic  for  animals  (cats),  whereas  the  amoeba 
dysenteriae  causes  dysentery  in  animals.  Whether  this 
difference  in  virulence  is  permanent,  or  whether  the  two 
amebae  are  identical,  the  one  merely  having  lost  its  viru- 
lence temporarily,  or  the  other  having  acquired  virulence 
temporarily,  can  not  be  decided  at  present  in  the  absence 
of  a  method  of  culture.  Quincke  observed  as  the  causa- 
tive agent  in  a  case  of  rather  mild  dysentery  of  autoch- 
thonous origin  in  Kiel  an  ameba  closely  resembling  that 
of  dysentery,  but  equally  nonpathogenic  for  cats  as  the 
intestinal  ameba.  He  places  this  amoeba  coli  mitis  between 
the  amoeba  intestini  vulgaris  and  the  ameba  of  dysentery 
(amoeba  coli  (Losch)).  (See  Fig.  83.)  Such  transitions 
render  it  probable  that  the  virulence  of  the  amebae  is  no 
more  constant  than  is  that  of  the  bacteria. 

Examination  of  the  Stools  for  Amebae. — In  examining 
the  stools  for  amebae  the  most  important  requirement  is 
that  the  stools  should  be  quite  fresh  and  be  examined  as 
soon  as  possible  after  evacuation.  It  is  best  to  receive  the 
stool  in  a  vessel  previously  heated  (in  water  at  a  tempera- 
ture of  40°  C. — 104°  F.).  If  the  stool  is  thin,  a  drop  is 
simply  placed  beneath  the  cover-glass  ;  when  blood-tinged 
flocculi  of  mucus  are  present,  these  are  examined.  If  the 
stool  is  mushy,  a  dilution  with  a  warm  solution  of  sodium 


372  CLINICAL  BACTERIOLOGY. 

chlorid  (from  35°  €.—95°  R— to  40°  €.—104°  R)  is 
made.  If  the  stool  is  formed,  only  the  superficial  layer  of 
mucus  is  examined.  To  fix  and  to  preserve  the  amebae,  a 
portion  of  the  material  containing  them  is  spread  in  as  thin 
a  layer  as  possible  upon  a  cover-slip,  which  is  at  once  in- 
troduced into  absolute  alcohol  before  it  dries.  The  amebae 
take  stains  less  well  than  the  bacteria  ;  so  that  in  cover- 
slip  preparations  they  are  distinguished  by  their  pallor. 
Methylene-blue  is  best  suited  for  staining.  The  nucleus 
stains  more  deeply  than  the  protoplasm.  More,  however, 
is  to  be  learned  from  examination  of  fresh  material  than 
from  that  of  stained  preparations,  and  if  the  observation 
cover  a  protracted  period,  a  warm  stage  is  a  useful  adjunct. 


MALARIA. 

The  exciting  agent  of  malaria  y^^,^  discovered  in  1880  by 
Laveran  in  the  blood  of  patients  suffering  from  that  dis- 
ease. 

The  parasites  of  7nalaria  are  unicellular  forms  of  life 
that  at  an  early  stage  are  endowed  with  ameboid  move- 
ment. They  belong  to  the  class  of  protozoa,  the  lowest 
forms  of  animal  life  ;  but  there  is  no  unanimity  of  opinion 
with  regard  to  their  position  in  the  zoologic  scale.  Metsch- 
nikoff  included  them  in  the  class  of  sporozoa,  and  con- 
sidered them  as  coccidia.  Others  placed  them  among 
gregarines,  Kruse  among  the  earliest,  and  he  designated 
them  as  hemogregarines.  Still  others  believe  them  to  be 
amebae,  and  include  them  among  the  rhizopods.  Mingazinni 
suggests  that  the  entire  group  of  parasites  that  invade  the 
red  blood-corpuscles  be  designated  hemosporidia.  In  con- 
sequence of  this  diversity  of  opinion  a  suitable  name  for  the 
parasites  of  malaria  is  wanting.  The  designation  malarial 
Plasmodia,  proposed  by  Marchiafava  and  Celli,  is  inappro- 
priate, as  Plasmodium  indicates  a  body  resulting  from  the 
confluence  of  numerous  amebae,  all  of  which  preserve  their 
nuclei. 

Morphology  and  Biology  of  the  Parasites  of  Mala- 
ria (Plate  I). — The  malarial  parasites  vary  in  size  from  one 
to  ten  microns  in  accordance  with  the  age  of  the  individual  cell. 
The  juvenile  forms  are  in  general  flattened  and  disc-like,  and 
their  shape  varies  with  their  ameboid  movement.     The  mature 


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Various  forms  of  malarial  parasites  (Thayer  and  Hewetson) :  Figs.  1  to  10,  in- 
clusive, tertian  organisms  ;  Figs.  11  to  17,  inclusive,  quartan  organisms;  Figs.  18  to 
27,  inclusive,  estivo-autumnal  organisms. 

Fig.  1. — Young  hyaline  form  ;  2,  hyaline  form  with  beginning  pigmentation  ; 
3,  pigmented  form  ;  4,  full-gx-own  pigmented  form  ;  5,  6,  7,  8,  segmenting  forms ;  9, 
extracellular  pigmented  form  ;  10,  flagellate  form. 

Fig.  11. — Young  hyaline  form;  12,  13,  pigmented  forms;  14,  fully-developed 
pigmented  form  ;  15,  16,  segmenting  forms  ;  17,  flagellate  form. 

Figs.  18,  19,  20.— Ring-like  and  cross-like  hyaline  forms;  21,  22,  pigmented 
forms ;  23,  24,  segmenting  forms  ;  25,  26,  27,  crescents. 


MALARIA.  373 

cells  are  spherical.  A  distinct  cell-membrane,  visible  as  a 
double  contour,  is  not  peculiar  to  most  varieties.  The  proto- 
plasmic body  is  relatively  small  in  the  juvenile  forms,  the 
nucleus  preponderating.  In  the  older  cells  these  conditions  are 
reversed.  In  the  living  parasites,  especially  at  an  early  period, 
nucleus  and  plasma  are  usually  not  differentiable.  In  stained 
preparations  (p.  393)  the  unstained  nucleus  is  clearly  distin- 
guishable from  the  deeply  stained  cell-body.  Differentiation 
into  an  outer  layer  of  cytoplasm  (ectoplasm)  and  a  central 
portion  (entoplasm)  is,  on  the  whole,  not  practicable.  The 
plasma  appears  mostly  homogeneous  and  hyaline,  although  ma- 
ture parasites  at  times  present  dense,  slightly  refractive  granula- 
tions. In  the  older  cells  the  substance  of  the  plasma  generally 
contains  from  a  brownish-red  to  a  black  pigment — the  malarial 
pigment,  or  melafiin,  which  is  considered  a  digestive  product  of 
hemoglobin.  This  assumes  the  form  in  part  of  fine,  dust-like 
particles,  in  part  of  coarser  granules;  or  it  appears  as  delicate 
needles  up  to  i  /t  in  length.'  The  pigment  often  exhibits  a 
peculiar  dancing  and  wriggling  movement,  which  only  resembles, 
but  is  not  identical  with,  the  Brownian  molecular  movement,  and 
is  believed  to  be  an  active  motor  procedure.  In  addition  to  the 
ameboid  movement  and  that  of  the  pigment  the  malarial  para- 
sites are  characterized  by  a  third  mode  of  motion — namely,  that 
dependent  upon  flagella.  From  the  fully  developed  round 
parasite  arise  flagella  that  move  actively,  in  part  become  free, 
and  then  retain  their  motility.  According  to  Kruse,  these 
structures  must  be  considered  as  evidences  of  degeneration.  In 
addition  to  pigment  the  cell-body  of  the  malarial  parasites  fre- 
quently contains  vacuoles,  mostly  small  and  few.  Often  difficult 
of  differentiation  from  these  is  the  relatively  large,  vesicular, 
generally  eccentric  nucleus,  which  in  the  living  cell  is  only 
recognizable  by  its  distinct  contour,  and  in  stained  preparations 
exhibits  no  nuclear .  membrane,  but  a  distinct  deep-black 
nucleolus  at  the  periphery.  The  nucleolus  is  often  surrounded  by 
a  feebly  stained  zone,  while  the  nucleus  itself  does  not  take  the 
stain.  Kruse,  upon  the  basis  of  his  investigations,  does  not 
admit  the  significance  of  this  large  nucleus,  but  he  considers  the 
nucleolus  as  the  nucleus. 

Laveran' s  Crescents. — Morphologically  distinct  from  these 
simple  forms  of  malarial  parasites  (corps  spheriques  of  Laveran) 
are  the  crescents  of  Laveran  (corps  en  croissant)  and  the  spher- 
ical and  spindle-shaped  bodies  related  to  them  (spheres  of  the 
crescentic  series).  The  crescents  have  the  shape  of  a  half- 
moon,  are  from  eight  to  ten  microns  long,  at  the  middle  from  two 
to  three  microns  thick,  and  are  characterized  by  a  considerable 
degree  of  refractive  power.  They  always  contain  pigment, 
usually  in  abundance,  but  sometimes  only  a  few  granules.     This 


374  CLINICAL   BACTERIOLOGY. 

is  usually  collected  at  the  center,  frequently  in  the  form  of  a 
figure  of  eight,  although  it  may  be  distributed  throughout  the 
entire  crescent ;  in  the  latter  event  granules  are  frequently  in 
tremulous  movement.  The  concentrated  pigment  is  invariably 
still.  The  crescentic  bodies  possess  a  membrane  (Mannaberg), 
which,  however,  is  not  visible  in  all  specimens.  At  times  their 
poles  are  united  on  the  concave  aspect  by  a  delicate  curved  line 
(remainder  of  the  host-cell).  The  crescents  are  not  endowed 
with  ameboid  movement ;  they  have,  however,  the  faculty  of 
slowly  changing  their  form  and  becoming  spindle-shaped,  oval, 
and  finally  quite  round  bodies.  This  transformation,  which  can 
be  observed  under  the  microscope,  takes  place  quite  gradually, 
in  the  course  often  of  several  hours.  In  fresh  blood-prepara- 
tions containing  numerous  malarial  parasites,  there  are  always 
present  a  few  spindles,  ovals,  and  spheres,  and  Mannaberg  is  of 
the  opinion  that  these  changes  in  the  shape  of  the  crescents  are 
dependent  exclusively,  or  almost  exclusively,  upon  the  removal 
of  the  parasite  from  the  human  body,  and  that  they  do  not 
occur  at  all,  or  but  exceptionally,  within  the  blood-vessels.  By  no 
means  all  crescents,  further,  undergo  the  changes  in  shape  de- 
scribed, but  the  larger  number  retain  their  shape  in  blood-prepara- 
tions preserved  in  a  moist  chamber.  In  the  sphere  resulting  from 
the  crescent  the  pigment  is  generally  arranged  in  the  shape  of  a 
wreath.  After  some  time  it  begins  to  move,  and,  with  tremu- 
lous movement,  to  intermingle,  and  soon  the  moving  granules 
of  melanin  occupy  the  entire  cell-body.  Flagella  also  then  form, 
and  the  entire  body  suddenly  engages  in  jerking  movements, 
while  at  the  periphery  projections  and  retractions  take  place. 
After  some  time  processes  resembling  in  form  the  finger  of  a  glove 
appear,  which  are  formed  by  the  membrane  of  the  body.  This 
membrane  ruptures,  the  processes  retract,  and  long,  slender  fila- 
ments are  thrust  out  from  them,  which  actively  lash  themselves 
about.  These  flagella  usually  present  a  bulbous  enlargement  at 
their  free  extremity,  and  they  often  exhibit  in  their  course  knob- 
like swellings  that  appear  to  change  place.  From  one  to  five 
filaments  develop  from  a  single  sphere.  Their  active  movement 
soon  diminishes,  and  has  entirely  disappeared  after  from  fifteen 
to  thirty  minutes.  The  resting  filaments  remain  attached  to  the 
body;  although  often  they  separate  and  then  move  actively 
about  free  in  the  blood.  The  membrane  ruptured  in  the  exit 
of  the  flagella  becomes  rolled  together  and  occupies  the  per- 
iphery of  the  sphere  in  the  form  of  a  globule  or  a  ringlet.  Such 
globules  distinguish  the  spheres  resulting  from  the  crescents  from 
the  other  spherical  elements  of  the  malarial  parasites  already 
described.  Further,  the  spherical  bodies  resulting  from  the 
crescents  have,  also  after  the  loss  of  their  membrane,  when, 
therefore  the  double  contour  is  no  longer  present,  still  a  much 


MALARIA.  375 

more  sharply  defined  boundary-line  than  the  other  spherical 
forms.  Some  crescents  undergo  segmentation,  usually  dividing 
transversely  through  the  middle  of  the  body. 

Relation  of  the  Parasites  to  the  Red  Blood-corpus- 
cles.— The  parasites  described  appear  in  part  free  in  the  blood- 
plasma,  and  in  part  in  association  with  the  red  blood-corpuscles. 
This  association  is  considered  by  Laveran  as  one  of  simple  adhe- 
sion of  the  parasites  to  the  blood-cell,  whereas  Marchiafava  and 
Celli,  in  opposition  to  this  extraglobular  mode  of  life,  assume 
an  actual  penetration  of  the  parasite  into  the  blood-cell,  an 
endoglobular  mode  of  life.  Both  views  are  probably  correct. 
The  smaller  forms  appear  impressed  into  the  surface  of  the  red 
blood-corpuscles,  to  which  they  are  merely  closely  attached, 
whereas  the  older  forms  certainly  occur  within  the  bodies 
of  the  blood-corpuscles.  This  latter  occurrence  can  be  observed 
especially  when  the  parasites  leave  the  infected  blood-corpuscle. 
In  this  process  the  residuum  of  the  blood-corpuscle  can  be  seen  to 
rupture,  while  its  contents,  stained  with  hemoglobin,  are  dis- 
tributed in  fine  drops  in  all  directions.  The  endoglobular 
arrangement  is  the  rule,  especially  for  the  crescents  of  Laveran, 
as  Marchiafava  and  Celli  have  demonstrated.  The  infected 
blood-corpuscles  are  usually  soon  decolorized,  while  the  para- 
sites at  the  same  time  take  up  pigment.  In  the  presence  of 
some  varieties  of  the  parasite  hypertrophy  of  the  infected  blood- 
corpuscle  is  observed  to  take  place,  while  others  cause  diminu- 
tion in  size  and  shrinking  of  the  blood-discs  in  which  they 
occur. 

Sporulation. — The  propagation  of  malarial  parasites  takes 
place  through  spores  that  form  in  the  mature  cells.  In  the 
fully  developed  parasite  a  greater  or  smaller  number  of  bodies 
develop,  each  of  which  exhibits  complete  cell-structure.  This 
process  of  sporulation  terminates  the  existence  of  the  spore-form- 
ing bodies ;  the  spores  disperse,  and  of  the  mother-cell  only  a 
dead  residual  body  remains,  consisting  mainly  of  the  pigment  of 
the  mother-parasite.  The  systematic  arrangement  of  the  spores 
within  the  sphere  around  a  mass  of  pigment  often  seated  cen- 
trally gives  rise  to  peculiar  appearances  that  have  been  compared 
with  those  of  daisies  or  sunflowers.  Every  spore  contains  a 
visible  nucleolus,  while  the  formation  of  the  nucleus  often  takes 
place  later. 

Development  of  the  Parasites. — According  to  Golgi,  in 
the  development  of  the  parasites  the  unpigmented  spore  moves 
about  free  for  a  time  in  the  blood-plasma,  and  meanwhile  pos- 
sibly grows  somewhat ;  it  then  becomes  attached  to  a  red  blood- 
corpuscle,  in  which  it  finds  the  conditions  for  its  further  devel- 
opment. 

The  young  parasite,  into  which  the  spore  within  the  blood- 


376  CLINICAL  BACTERIOLOGY. 

corpuscle  has,  without  especial  external  alteration,  been  trans- 
formed, grows  and  deprives  the  red  blood-corpuscle  of  nutritive 
material.  It  converts  the  hemoglobin  of  the  host  into  melanin, 
which  it  collects  in  the  outer  layer  of  its  protoplasmic  body.  It 
grows  until  it  reaches  the  height  of  its  development,  when  it 
sometimes  entirely  fills  the  red  blood-corpuscle,  and  it  then  gives 
rise  to  new  spores,  in  the  setting  free  of  which  the  residuum  of 
the  blood-cell  is  ruptured. 

With  regard  to  the  relation  of  the  crescentic  bodies  to  this 
process  of  development  of  the  parasites,  it  is  commonly  assumed 
that  the  crescents  result  from  the  small  ameboid  parasites,  of 
which  Laveran  considers  them  to  be  the  encysted  form,  while 
Councilman  believes  them  to  be  their  spores.  Mannaberg  con- 
siders the  crescents  as  copulation-bodies  (syzygia),  which  result 
from  the  union  of  two  ameboid  malarial  parasites.  The  two 
parasites  do  not  completely  coalesce  (pseudo-conjugation),  and 
they  may  subsequently  separate  (segmentation).  Kruse believes 
the  crescentic  bodies  to  be  no  longer  capable  of  infection,  but  he 
considers  them  the  harmless  residue  of  the  infectious  process. 

Polymorphism  or  Multiplicity  of  the  Malarial  Para- 
sites.— The  appearances  presented  by  the  blood  in  the  dif- 
ferent types  of  malarial  fever  are  most  variable,  and  for  a 
long  time  there  has  been  a  difference  of  opinion  as  to 
whether  there  are  various  forms  of  malarial  parasites,  with 
a  special  species  of  parasite  for  every  type  of  fever,  or 
whether  the  various  forms  of  the  parasite  are  but  different 
phases  of  the  same  organism.  Laveran  and  his  adherents 
consider  the  parasites  as  polymorphous.  They  believe 
that  the  various  types  of  fever  are  induced  not  by  different 
species  of  parasites,  but  as  a  result  of  variations  in  the 
predisposition  of  the  organism  attacked.  The  Italian 
school,  on  the  other  hand,  considers  the  exciting  agents  of 
the  various  types  of  fever  as  belonging  to  different  species. 
Golgi  makes  three  varieties  :  the  parasites  of  quartan,  those 
of  tertian,  and  those  of  irregular,  febrile  type.  According  to 
his  view,  the  quotidian  type  is  dependent  upon  either  two 
generations  of  parasites  of  the  tertian  or  three  generations 
of  the  quartan  type,  of  which  each  generation  is  separated 
in  its  development  from  the  other  by  a  period  of  twenty- 
four  hours.  Golgi  has  described  the  cycle  of  development 
for  these  three  varieties.  Marchiafava  and  Celli  recognize 
Golgi's  tertian  and  quartan  parasites.  As  the  exciting 
agent  of  the  irregular  fever,  however,  they  consider  the 
small  ameboid  forms  from  which  the  crescents  are  derived. 


MALARIA.  377 

At  times  these  also  give  rise  to  typical  quotidian  fever. 
The  different  parasites  appear  to  Marchiafava  and  Celli, 
however,  as  varieties  of  a  single  parasite.  Grassi  and 
Feletti  have  gone  so  far  as  to  describe  five  species. 

As  cultivation  of  the  malarial  parasites  is  at  present  not 
possible,  the  question  whether  the  forms  of  parasites  are 
constant  and  variable,  or  whether  each  form  gives  rise  to  a 
definite  type  of  fever,  can  only  be  determined  by  experi- 
mental conveyance  of  the  parasites  with  the  blood  of 
malarial  patients.  Such  inoculation- experiments  have  been 
repeatedly  made  in  human  beings.  The  results,  as  sum- 
marized by  Mannaberg,  are  as  follows  :  Of  sixteen  care- 
fully performed  experiments,  there  was  in  fourteen  a  com- 
plete concurrence  between  the  forms  of  parasites  from  the 
source  of  inoculation  and  those  from  the  inoculated  person  ; 
whereas  in  two  cases  the  inoculated  person  exhibited  other 
forms  than  those  from  the  source  of  inoculation.  The  two 
cases,  however,  that  appear  to  support  the  view  of  poly- 
morphism are,  on  more  careful  scrutiny,  as  Mannaberg 
shows,  not  entirely  free  from  criticism,  so  that  the  results 
of  the  sixteen  experiments  render  it  highly  probable  that 
the  individual  varieties  of  parasites  represent  distinct  spe- 
cies, which  do  not  undergo  a  transformation  into  other 
varieties.  From  the  experiments,  the  conclusion  is  to  be 
drawn  further  that  an  unmistakable  relation  exists  between 
the  type  of  fever  and  the  species  of  parasites. 

Classification  of  the  Malarial  Parasites. — The  funda- 
mental types  of  intermittent  fever  are  the  quotidian,  the 
tertian,  and  the  quartan.  In  addition  there  occur  fre- 
quently irregular X-y^^s,  which  pursue  their  course  in  part 
continuously,  in  part  with  distinct  remissions,  or  with 
attacks  following  one  immediately  upon  another.  Of  the 
quotidian  variety  two  forms  have  long  been  distinguished — 
namely,  the  double  tertian  and  the  triple  quartan.  The 
different  types  are  dependent  upon  different  forms  of  para- 
sites.    The  following  are  among  those  best  known  : 

I.  The  Quartan  Parasite. — The  juvenile  form  of  the 
quartan  parasite  is  an  unpigmented  body,  with  slow,  ame- 
boid movement,  visible  as  a  small,  bright  spot  within  the 
infected  blood-corpuscle.  It  grows  but  little  during  the 
first  twelve  or  twenty-four  hours.  Then  pigment  appears 
in  the  outer  layer  in  the  form  of  coarse  granules  and  rods  ; 
this  is  nonmotile.     With  increasing  pigmentation  the  para- 


378  CLINICAL  BACTERIOLOGY. 

site  loses  its  previous  torpid  motility,  becoming  spherical 
and  filling  the  blood-corpuscle  one-third  or  one-half.  It 
then  continues  to  grow  slowly  until  it  has  attained  the  full 
size  of  the  blood-corpuscle,  so  that  no  portion  of  this 
remains  visible,  and  the  parasite  appears  to  be  free.  Sporu- 
lation  now  takes  place,  the  pigment-granules  concentrating 
at  the  center,  while  a  radiate  striation  makes  its  appear- 
ance in  the  plasma,  first  at  the  periphery,  subsequently 
also  at  the  center,  gradually  becoming  more  distinct  and 
dividing  the  parasite  into  from  eight  to  twelve  segments. 
These  segments  separate  more  distinctly  from  one  another 
as  oval  bodies  (daisy-form),  and  each  contains  a  bright 
spot — the  nucleolus.  Spore -formation  is  now  completed, 
and  the  spores  are  set  free  by  rupture  of  the  mother-cell. 
The  entire  process  of  development  has  occupied  seventy- 
two  hours.  Segmentation  takes  place  before  and  during 
the  febrile  attack.  About  three  hours  before  the  occur- 
rence of  the  chill  the  first  complete  sporulation-bodies  are 
visible  in  the  blood.  The  red  blood-corpuscles  that  have 
been  infected  with  quartan  parasites  do  not  undergo  change 
in  size.  The  parasites  at  times  progress  to  sporulation 
before  they  have  attained  the  size  of  the  blood-corpuscle. 
They  then  generally  give  rise  to  from  four  to  six  spores. 
The  extrusion  of  flagella  is  observed  seldom  and  only  in 
the  younger  forms. 

In  the  regular  progress  of  development  of  the  quartan 
parasites  it  is  relatively  easy  to  distinguish  several  genera- 
tions from  one  another  when  these  are  present. 

The    quartan    parasite    induces    typical    quartan    fever 

(looiooiooi) ;  two  or  three  generations  separated  by  an 
interval   of  twenty-four  hours  give   rise  to  double  quartan 

(120 1 20 1 20)    or  triple   quartan  (123  123  123).     The  last 

may  be  considered  a  false  quartan.  If  several  generations 
of  the  quartan  parasite  are  present  whose  development  is 
separated  not  by  intervals  of  twenty-four  hours,  but  by 
shorter  or  longer  intervals,  irregular  types  of  fever  re- 
sult. 

2.  The  tertian  parasite  completes  its  cycle  of  develop- 
ment within  forty-eight  hours.  The  juvenile  form  re- 
sembles that  of  the  quartan  parasite  :  presenting  a  small 
plasmic  body,  with  a  nucleus  free  from  pigment,  in  the  living 


MALARIA.  379 

state  visible  only  as  a  bright  spot  within  a  red  blood-cor- 
puscle. It  is  actively  motile,  and  extrudes  numerous  pseu- 
dopods.  In  the  first  twenty-four  hours  (first  phase  of 
Golgi)  it  develops  gradually,  and  collects  finely  granular 
pigment  within  itself,  which  continues  in  active  vibratile 
movement,  with  a  preference  for  the  outer  layer  of  the 
plasmic  body.  With  increasing  deposition  of  pigment  the 
activity  of  ameboid  movement  diminishes,  without,  however, 
ceasing  entirely.  After  twenty -four  hours  the  parasite  has 
attained  about  half  the  size  of  the  red  blood-corpuscle,  which 
itself  has  lost  in  color  and  has  undergone  increase  in  size — 
often  to  a  considerable  degree.  "  The  blood-corpuscles  in- 
fected with  tertian  parasites  are  frequently  distended  and 
chlorotic."  After  forty -eight  hours,  when  the  parasite  has 
attained  almost  the  size  of  the  blood-corpuscle  and  is "  no 
longer  in  motion  and  the  pigment  also  is  at  rest,  sporula- 
tion  takes  place.  Usually,  the  pigment  moves  again  toward 
the  center,  the  plasma  divides  into  from  fifteen  to  twenty 
round,  highly  refractive  globules,  which  sometimes  ar- 
range themselves  in  two  concentric  rows  (Golgi's  sun- 
flowers), but  frequently  lie  together  in  the  shape  of  berries, 
rosets,  or  grapes.  The  round  globules  are  smaller  than 
the  spores  of  the  quartan  parasites.  The  nucleolus  is  only 
with  difficulty  to  be  distinguished  in  them.  The  spores 
then  become  free,  and  after  a  time  infect  new  blood-cor- 
puscles, in  turn  themselves  to  repeat  the  same  process  of 
development.  By  no  means  all  parasites  undergo  sporula- 
tion,  just  as  is  the  case  with  the  quartan  parasite.  A  large 
number  of  tertian  parasites  remain  sterile.  These  barren 
elements  generally  become  as  large  as  the  parasites  that 
undergo  propagation,  or  larger.  In  them,  however,  the 
pigment  remains  actively  motile.  Laveran  considers  them 
as  hydropic  and  involved  in  degeneration.  They  may  still 
be  visible  in  the  blood  for  hours  after  the  attack,  and  even 
on  the  afebrile  days.  The  act  of  sporulation  corresponds 
also  in  tertian  fever  with  the  febrile  paroxysm.  Golgi  has 
shown  that  as  early  as  three  hours  before  the  chill  the 
temperature  begins  to  rise,  and  that  then  the  first  spores 
make  their  appearance  in  the  blood.  They  are,  however, 
most  numerous  at  the  time,  of  the  chill.  In  the  mature 
tertian  parasites  the  formation  of  flagella  can  often  be  ob- 
served. Kruse  considers  this  process,  likewise,  as  evidence 
of  degeneration. 


380  CLINICAL  BACTERIOLOGY. 

The  tertian  parasite  causes  typical  tertian  (qyqy  (^loioioi). 

Two  generations  of  the  parasite  may  give  rise  to  a  false 

quotidian,  a  so-called    double  tertian  (12 12 121).      Several 

generations  not  separated  from  one  another  by  intervals  of 
twenty-four  hours  give  rise  to  irregular  fever. 

J.  The  Quotidian  Parasite. — The  commencement  of  the 
cycle  of  development,  which  in  its  entirety  occupies  twenty- 
four  hours,  is  like  that  of  the  other  parasites.  The  juvenile 
form,  unpigmented,  consisting  of  plasmic  body  and  nucleus, 
infects  the  red  blood-corpuscle.  The  parasite  is  actively 
motile,  and  is  often  recognized  by  this  property,  while  by 
reason  of  its  extremely  delicate  contour  and  its  color,  which 
is  only  slightly  less  pale  than  that  of  the  red  blood-corpus- 
cle, it  is  scarcely  distinguishable  from  this.  On  removal  of 
the  blood  the  parasite  soon  loses  its  motility,  at  the  latest 
after  the  lapse  of  an  hour.  A  whitish  ring  is  then  seen  to 
form,  with  a  reddish  center.  In  consequence  of  a  pro- 
jection at  one  point  of  the  periphery  the  ring  frequently 
resembles  a  seal-ring.  Mannaberg  believes  that  these 
forms  are  only  closely  attached  to  the  red  blood-corpuscles, 
and  are  not  contained  within  them.  The  red  spot  in  the 
center  is  thought  to  depend  upon  dilution  of  the  plasma  and 
the  appearance  of  the  underlying  red  blood-corpuscle  as 
seen  through  this.  The  annular  form  may  return  to  the 
ameboid.  The  ameboid  parasite  does  not  grow  much — alto- 
gether to  only  about  one-third  the  size  of  the  red  blood- 
cell.  At  the  same  time  a  fine  pigment,  which  is  often  only 
reddish  and  is  but  slightly  motile,  collects  at  the  periphery. 
After  the  lapse  of  twenty-four  hours  the  pigment  becomes 
concentrated  at  the  center  or  at  the  periphery  in  the  form 
of  dark,  resting  clumps,  and  the  parasite  disintegrates  within 
the. red  blood-corpuscle  into  the  smallest  spores  (from  five 
to  ten).  This  process  of  sporulation  takes  place,  according 
to  Marchiafava  and  Celli,  only  in  the  internal  viscera  of  the 
body,  and  scarcely  at  all  in  the  peripheral  blood.  For  this 
reason  the  spores  are  encountered  in  abundance  in  the 
blood  obtained  from  the  spleen,  whereas  they  are  found  not 
at  all,  or  only  isolated,  in  blood,  from  the  finger-tip.  The 
red  blood-cells  infected  with  the  quotidian  parasites  con- 
tract and  become  brass-colored  (brassy  bodies.)  After  the 
quotidian  parasite  has  been  present  in  the  blood  for  several 
days  the  crescents  already  described  invariably  appear,  with 


MALARIA.  381 

their  secondary  form,  the  spindle-shaped  (cigar-shaped) 
bodies  and  the  spheres.  With  regard  to  the  nature  of  the 
connection  between  these  forms  and  the  ameboid  parasite 
there  is  as  yet  no  unanimity  of  opinion.  The  various  views 
with  regard  to  the  source  and  the  destiny  of  the  crescents 
have  already  been  briefly  outlined  (p.  376). 

The  quotidian  parasite  gives  rise  to  typical  quotidian 
fever,  and,  when  present  in  several  generations,  to  continued, 
or  irregular,  fever.  The  fever  induced  by  the  quotidian 
parasite  differs  clinically  from  that  due  to  the  tertian  and 
quartan  parasites  in  its  malignancy.  It  recurs  obstinately, 
and  often  gives  rise  to  profound  anemia,  and  to  other  per- 
nicious manifestations  (diarrhea,  cachexia,  coma,  etc.).  The 
recurrences  take  place  usually  from  seven  to  fourteen  days 
after  the  first  febrile  paroxysm.  The  crescents  are  generally 
held  responsible  for  the  recurrences.  These  parasites  are 
present  in  the  blood  in  the  afebrile  interval,  and  are  believed 
to  be  capable,  through  segmentation  or  true  spore -formation, 
of  giving  rise  to  new  ameboid  forms.  The  new  paroxysms 
would,  thus,  not  be  true  recurrences,  but,  as  Golgi  believes, 
only  the  expression  of  a  type  with  long  intervals.  This  is 
denied  by  others,  and  the  crescents  are  considered  to  be 
only  degenerative  forms  that  are  incapable  of  contributing 
further  to  the  formation  of  new  individuals.  It  is  true  that 
with  the  presence  exclusively  of  crescents  and  their  spheres 
in  the  blood,  fever  is  generally  not  present.  On  the  other 
hand,  the  demonstration  of  these  bodies  in  the  blood  indi- 
cates with  certainty  that  fever  existed  a  short  while  previ- 
ously, and  with  great  probability  that  new  paroxysms  will 
occur  within  a  short  time. 

It  is  to  be  mentioned,  finally,  that  there  is  an  unpig- 
mented  quotidian  parasite  (Marchiafava  and  Celli),  which  is 
distinguished  from  the  ordinary  quotidian  parasite  only 
by  the  complete  absence  of  pigment.  This  parasite,  like- 
wise, forms  crescents,  but  these  are  unsupplied  with  pig- 
ment. The  clinical  course  of  the  infection  with  these 
unpigmented  parasites  is  in  no  wise  different  from  that  de- 
scribed for  infection  with  the  pigmented  quotidian  para- 
site. 

^.  The  malignant  tertian  parasite  has  been  separated  by 
Marchiafava  and  Bignami  as  a  special  species.  It  stands 
close  to  the  quotidian  parasite,  but  is  believed  to  differ 
from  this  in  the  fact  that  its  cycle  of  development  occupies 


382  CLINICAL  BACTERIOLOGY. 

forty-eight  hours,  that  it  becomes  somewhat  larger  (at  the 
time  of  sporulation  it  is  one-half  or  two-thirds  the  size  of 
the  blood-corpuscle),  and  that  it  remains  unpigmented  for 
twenty-four  hours  and  then  begins  to  acquire  pigment, 
without,  however,  losing  its  motihty.  This  parasite  also 
forms  annular  bodies,  and  especially  crescents.  It  gener- 
ally develops  from  eight  to  fifteen  spores.  The  blood- 
corpuscles  infected  by  it  become  brass-colored,  and  usually 
shrink,  but  never  increase  in  size.  The  parasite  is  distin- 
guished by  these  last-named  peculiarities  from  the  ordinary 
tertian  parasite  (Golgi),  which,  besides,  in  all  correspond- 
ing stages  is  larger,  is  provided  with  more  pigment,  and 
forms  larger  spores  (from  fourteen  to  twenty).  The  malig- 
nant tertian  parasite  gives  rise  to  severe  tertian  types  of 
fever,  and  also  to  peculiar  temperature-curves^  with  quite 
short  afebrile  intervals  often  lasting  only  a  few  hours,  and 
with  regular  pseudo-crises,  and,  finally,  continued,  or  ir- 
regular, fever.  All  of  the  varieties  of  fever  induced  by  it 
are  characterized  by  the  malignancy  peculiar  also  to  the 
quotidian  parasite. 

Mixed  Infection. — In  a  number  of  cases  of  intermittent 
fever  several  of  the  parasites  described  are  found  together 
in  the  blood,  and  with  especial  frequency  pigmented  quo- 
tidian parasites,  with  unpigmented,  and  not  rarely  also 
quartan  with  tertian  parasites.  Golgi  found  in  one  patient 
three  generations  of  the  quartan  parasite  and  two  of  the 
tertian  parasite.  All  of  the  various  components  may  find 
definite  expression  in  the  temperature-curve,  or  the  fever 
may  pursue  an  irregular  course.  It  may  be,  further,  that 
the  temperature-course  corresponds  with  only  one  variety 
of  parasite,  while  the  other  appears  to  exert  no  influence. 

Diagnosis  of  the  Malarial  Parasites  (Method  of 
Blood-examination. — A  drop  of  blood  is  taken,  as  in 
ordinary  examination,  from  the  lobule  of  the  ear  or  the  tip 
of  the  finger,  after  previous  cleansing  by  means  of  brush, 
soap,  mercuric  chlorid,  alcohol,  and  ether.  Slides  and 
cover-glasses  must  be  cleansed  (with  alcohol  and  ether)  and 
dried  with  especial  care.  It  is  advisable  not  to  manipulate 
them  with  the  fingers  at  all,  but  to  grasp  them  with  the  aid 
of  suitable  forceps  (Ehrlich).  A  puncture  is  made  with 
a  lancet,  the  extruded  drop  of  blood  is  received  upon  the 
cover-slip,  and  this  is  inverted  simply  upon  the  slide.  It 
is  especially  important  that  the  drop  obtained  be  not  too 


MALARIA.  383 

large.  The  blood-cells  must  lie  side  by  side  somewhat 
scattered,  as  arrangement  in  rouleaux  would  conceal  the 
parasites.  The  preparation  is  then  examined  with  a  good 
oil-immersion  lens.  If  the  examination  is  to  be  continued 
for  some  time,  evaporation  is  prevented  by  surrounding  the 
margin  of  the  cover-slip  with  wax,  or  the  uniformly  dis- 
tributed drop  of  blood  is  examined  on  an  excavated  sHde, 
at  the  bottom  of  which  is  a  drop  of  water.  To  stimulate 
ameboid  movement,  a  warm  stage  should  be  employed. 
Examination  in  a  warm  room  is  possible,  however,  for  an 
hour  or  more,  without  the  aid  of  this  device. 

In  the  preparation  of  dry  specimens  also  it  is  important  to 
obtain  quite  a  small  drop  of  blood.  The  cover-slip  is  then 
quickly  drawn  over  the  surface  of  a  second  cover-slip,  and 
both,  protected  from  dust,  are  permitted  to  dry  in  the  air. 
The  dried  preparations  are  placed  for  fixation  for  from 
five  to  thirty  minutes  in  a  mixture  of  equal  parts  of  abso- 
lute alcohol  and  ether.  They  are  now  dried  between  lay- 
ers of  bibulous  paper,  and  are  stained — either  in  dilute 
aqueous  solution  of  methylene-blue,  and  then,  after  rinsing 
with  water,  in  two  per  cent,  alcoholic  (sixty  per  cent.)  solu- 
tion of  eosin ;  or  the  specimen  is  exposed  to  the  action  of 
both  stains  at  the  same  time.  For  this  purpose  Plehn 
recommends  the  following  mixture  : 

Coi;icentrated  aqueous  solution  of  methylene- 
blue,      60 

One-half  per  cent,  solution  of  eosin  (in  sev- 
enty-five per  cent,  alcohol), 20 

Distilled  water, 40. 

The  preparation  is  kept  in  this  solution  for  from  five  to 
ten  minutes,  and  is  then  rinsed  in  water,  dried,  and  examined 
in  xylol  Canada  balsam.  The  hemoglobin-containing  blood- 
cells  are  thereby  stained  red  ;  the  plasmic  body  of  the  para- 
sites, more  or  less  deeply  blue.  For  .rapid  examination, 
simple  treatment  with  aqueous  methylene-blue  suffices. 
For  special  examinations  and  for  the  determination  of  the 
finer  structural  relations,  a  large  number  of  methods  of 
fixation  and  staining  have  been  proposed.  For  diagnostic 
purposes,  examination  of  fresh  specimens  and  of  stained 
preparations  by  the  method  described  is  quite  sufficient. 

With  regard  to  the  diagnostic  utility  of  the  results  of 
the  examination,  the  following  statements  may  be  made : 


384  CLINICAL  BACTERIOLOGY. 

The  presence  of  even  a  single  undoubted  malarial  parasite 
establishes  the  diagnosis  with  certainty.  Should  the  result 
be  negative,  it  must  be  borne  in  mind  that  after  the  action 
of  quinin  the  parasites  at  times  can  not  be  found  in 
the  blood  ;  further,  that  they  are  sometimes  wanting  after 
quite  recent  infection  during  the  first  days  of  the  disease. 
Negative  results  from  a  single  observation  are,  therefore,  not 
conclusive,  even  when  a  large  number  of  specimens  have 
been  examined.  Repeated  examinations  must  be  made, 
preferably  from  three  to  ten  hours  in  advance  of  the  par- 
oxysm, when  the  parasites  have  reached  the  height  of  their 
development. 

In  the  determination  of  the  variety  of  parasites,  and 
with  regard  to  the  prognosis,  it  is  especially  important  to 
decide  whether  crescents  and  their  spindles  or  spheres  are 
present.  The  characteristics  of  these  have  been  described. 
The  recognition  of  the  species,  as  well  as  the  decision 
whether  several  generations  of  the  parasite  are  present, 
naturally  demands  continued  observations,  to  be  repeated 
in  the  course  of  several  hours.  The  differentiation  of  free 
bodies  moving  about  in  the  blood  is  attended  with  consider- 
able difficulty,  and  opportunity  is  here  afforded  for  confusion 
from  several  sources.  The  parasitic  nature  of  the  bodies 
contained  within  the  red  blood-corpuscles  is,  however,  more 
readily  appreciated.  The  presence  of  pigment  is  here 
especially  of  significance.  Only  the  unpigmented  juvenile 
forms  may  occasion  difficulty,  as  they  may  be  readily  mis- 
taken in  living  preparations  for  vacuoles  of  the  red  blood- 
corpuscles.  The  structure,  which  can  not  be  overlooked, 
especially  in  stained  preparations  (nucleus),  is  decisive  in 
this  connection  ;  the  vacuoles  of  the  blood-discs  are  struc- 
tureless. 

Explanation  of  the  Symptoms  of  Malaria  by  the 
Presence  of  the  Parasites. — The  parasites  of  malaria  are 
present  exclusively  in  the  blood.  Even  in  the  viscera  they 
are  found  only  within  the  capillaries.  The  melanemia  of 
malarial  patients,  which  has  long  been  known,  is  explained 
by  the  conversion  of  the  hemoglobin  into  melanin  within 
the  parasites.  The  melanin  is  set  free  in  the  process  of 
sporulation,  when  it  floats  about  in  the  plasma  and  is  taken 
up  by  the  leukocytes.  The  infection  of  the  blood-corpus- 
cles by  the  parasites  furnishes  a  direct  explanation  for  the 
anemia  that  arises  in  the  course  of  malaria.     The  infected 


MALARIA.  385 

blood-discs  are  destroyed  in  the  process  of  development  of 
the  parasites.  At  the  same  time  an  injurious  influence  is 
exerted  upon  the  uninfected  blood-corpuscles  by  a  parasitic 
poison  dissolved  in  the  blood-plasma.  A  chemic  toxic 
action  on  the  part  of  the  parasites  has,  it  is  true,  not  yet 
been  established.  It  is,  however,  rendered  probable  by  the 
fact  that  the  urine  and  the  sweat  of  malarial  patients,  both 
of  which  secretions  do  not  contain  the  parasite  itself,  are 
poisonous  to  rabbits,  and  are  capable  of  causing  death  in 
these  animals.  The  presence  of  a  poison  is  also  necessary 
for  the  explanation  of  the  febrile  attack.  The  paroxysm 
always  sets  in  with  sporulation — that  is,  with  disintegration 
of  the  completely  developed  parasite.  It  is  generally  as- 
sumed that  in  this  process,  simultaneously  with  the  spores, 
a  poison  is  set  free  that  is  thrown  into  the  blood  and  gives 
rise  to  the  fever.  Malaria  would  thus  be  a  form  of  proto- 
zoan septicemia  presenting  analogies  with  ordinary  bacte- 
rial septicemia.  The  toxic  action  would  then  without  diffi- 
culty explain  also  the  other  symptoms — the  diarrhea,  the 
dyspnea,  the  ecchymoses,  and  especially  the  nervous  symp- 
toms. The  bone-pains  are  usually  attributed  to  the  increased 
demands  upon  the  blood-forming  activity  of  the  bone- 
marrow,  and  comparable  with  those  observed  in  leukemia. 
Coma  may  be  induced  by  the  occlusion  of  the  cerebral 
vessels  with  the  parasites  themselves,  and  this  has  been 
demonstrated  microscopically  in  a  number  of  cases. 

Mode  of  Infection  with  Malaria. — Cultivation  of  the 
malarial  parasites  has  thus  far  not  been  successful.  Trans- 
mission of  the  disease  to  animals  has  likewise  not  been 
successful,  although  it  has  been  attempted  with  numerous 
varieties.  The  only  positive  result  that  has  been  obtained 
'in  this  connection  consists  in  the  survival  for  forty-eight 
hours  of  the  parasites  within  the  bodies  of  leeches  that 
had  been  applied  to  malarial  patients  (Rosenbach).  The 
malarial  parasites  are  known  only  as  blood-parasites  of 
human  beings.  No  conception  has  hitherto  been  possible 
as  to  where  and  in  what  form  they  exist  outside  of  the 
human  body.  For  this  reason  existing  knowledge  of  the 
manner  in  which  malarial  infection  takes  place,  and  which 
is  mainly  based  upon  empiric  observation,  is  not  very  ex- 
tensive. Malaria  may  be  transmitted  from  one  human 
being  to  another  by  means  of  the  blood.  Gerhardt  was 
the  first  to  demonstrate  this  by  subcutaneous  injections  of 
25 


386  CLINICAL  BACTERIOLOGY. 

malarial  blood.  Subsequently,  infection  was  repeatedly- 
transmitted  with  the  blood  of  malarial  patients  by  subcuta- 
neous and  intravenous  injection.  Malaria  is,  however,  not 
a  contagious  disease.  It  does  not  pass  from  one  human 
being  to  another  under  natural  conditions.  This  could  only 
be  possible  if  the  blood  of  a  malarial  patient  gained  en- 
trance into  the  body  of  another  individualj  and  probably 
few  opportunities  for  this  are  afforded.  The  parasites  are 
not  present  in  the  secretions  and  excretions  of  malarial 
patients  ;  they  appear  to  be  present  in  the  contents  of  the 
herpetic  vesicles  that  malarial  patients  often  exhibit.  At 
least,  malaria  has  been  inoculated  by  means  of  the  contents 
of  such  vesicles. 

The  malarial  parasites  must,  however,  be  present  some- 
where in  nature.  They  must  live  in  some  form  in  air,  earth, 
or  water,  in  certain  swampy  regions  in  which  the  disease 
is  endemic,  and  at  certain  times  becomes  epidemic.  The 
view  is  generally  held  that  the  parasites  are  inhaled  ;  some 
believe  that  they  are  taken  up  with  the  drinking-water. 
Transmission  through  the  bites  of  insects  is  theoretically 
possible,  and  is  admitted  by  some  observers,  but  it  is  not 
yet  demonstrated.* 

The  period  of  incubation  is  in  most  cases  from  eight  to 
fourteen  days  ;  cases  are,  however,  known  in  which  the 
disease  appeared  months  after  infection,  and  others  in  which 
the  period  of  incubation  was  only  one  or  two  days,  or  even 
hours.  These  differences  may  be  explained  by  the  num- 
ber of  the  infecting  germs,  and  by  the  varying  individual 
predisposition  of  those  infected.  That  the  germ  is  taken 
up  in  a  form  that  must  pass  through  certain  variations 
within  the  body  in  order  actually  to  become  the  malarial 
parasite  is  negatived  by  the  cases  with  a  short  period  of 
incubation. 

The  Action  of  Quinin  and  Spontaneous  Recovery  from 
Malaria. — Laveran  has  determined  that  addition  of  even  a 
very  dilute  solution  of  quinin  to  a  blood-preparation  at 
once  causes  cessation  of  the  ameboid  movements  of  the 
parasites.  Within  the  human  body,  also,  the  parasites  un- 
dergo visible  alterations  after  administration  of  quinin  :  they 
suffer  in  motility  and  undergo  degeneration,  and,  in  many, 
sporulation  does  not  take   place   in   the   normal   manner. 

*  Recent  observations  have  demonstrated  most  conclusively  that  malaria  is 
transmitted  by  some  varieties  of  mosquitoes. — A.  A.  E. 


LEYDENIA   GEMMIPARA   SCHAUDINN.  387 

According  to  Golgi,  the  spores  are  most  susceptible  to 
quinin,  and  the  mature  forms,  before  the  beginning  of  the 
process  of  segmentation,  are  somewhat  less  so,  and  the  en- 
doglobular,  juvenile  forms  still  less  so.  The  crescentic 
bodies  are  considered  as  entirely  insensitive  to  the  action 
of  quinin.  The  parasites  altered  by  the  action  of  quinin 
are  designated  as  **  quinin-varieties."  It  is  generally  ac- 
cepted that  quinin  is  a  specific  fatal  poison  for  the  malarial 
parasites  (Binz).  Quinin  certainly  exerts  a  curative  effect, 
and,  according  to  some  observers,  given  in  small  doses,  it 
is  also  of  prophylactic  utility. 

In  explanation  of  spontaneous  recovery  from  malaria 
Metschnikoff  considers  phagocytosis  a  most  important 
factor.  Probably  this  plays  a  not  insignificant  role,  but 
other  influences,  besides,  must  certainly  be  taken  into  con- 
sideration. As  has  been  mentioned,  in  every  case  of  malaria 
a  number  of  normally  developed  parasites  regularly  do 
not  undergo  sporulation.  The  remaining  sterile  elements 
are  still  visible  in  the  blood  for  from  twenty-four  to  forty- 
eight  hours,  when  they  undergo  degeneration.  Upon  what 
this  sterility  depends  is  not  known.  It  may,  however, 
readily  contribute  to  the  process  of  recovery,  should  a 
considerable  number  of  the  parasites  not  undergo  propa- 
gation. Further,  the  fever  itself,  as  in  the  bacterial 
diseases,  is  believed  to  exert  an  injurious  influence  upon 
the  parasites. 


LEYDENIA  GEMMIPARA  SCHAUDINN. 

In  connection  with  the  protozoa  of  dysentery  and  of 
malaria  mention  must  be  made  of  an  observation  by 
V.  Leyden,  who  found  an  organism  in  the  sterile  ascites 
fluid  obtained  from  two  patients  suffering  from  carcinoma. 
This  organism  is  an  ameba^  which  the  discoverer  investi- 
gated in  conjunction  with  the  zoologist  Schaudinn.  It  is 
spherical  or  irregular  in  shape,  and  from  3  to  36  //  long.  In 
hot  weather  it  retains  its  motility  for  four  or  five  hours  with- 
out the  employment  of  a  warm  stage.  The  ectoplasm  sends 
out  pseudopods,  in  whose  formation  the  entoplasm  also 
soon  takes  part.  The  latter  possesses  a  honeycomb  struc- 
ture, and  contains  yellowish,  refractive  granules,  perhaps 
hemoglobin  derived  from  the  inclosure  of  red  blood-cor- 


388  .  CLINICAL  BACTERIOLOGY. 

puscles.  Within  the  entoplasm  of  the  processes  vacuoles, 
food-residua,  and  excrementitious  granules  resembling  crys- 
tals are  also  found.  Among  the  vacuoles  one  exhibits  pul- 
sation, undergoing  contraction  about  every  fifteen  minutes. 
The  nucleus  is  about  one-fifth  as  large  as  the  resting  ameba. 
The  pseudopods  of  adjacent  bodies  frequently  unite,  and 
there  thus  result  plasmodia  consisting  of  as  many  as  forty 
segments.  Multiplication  takes  place  through  division  or 
budding.  Leydenia  belongs  to  the  rhizopods.  Von  Leyden 
expresses  himself  with  reserve  in  regard  to  its  etiologic 
significance. 


APPENDIX. 


L  BACTERIOLOGIC  EXAMINATION  OF  SOIL, 
AIR,  AND  WATER. 

Although  air  and  soil  do  not  play  the  important  role  in 
the  transmission  of  disease  assigned  to  them  by  the  path- 
ology of  the  past  (pp.  169,  191),  they  nevertheless  fre- 
quently act  as  media  for  the  transmission  of  disease-germs, 
as  has  been  shown  in  the  preceding  chapters.  Bacterio- 
scopic  examination  of  air  and  soil  is  frequently  demanded 
of  the  hygienist  when  it  is  desired  to  determfne  the  avail- 
ability of  ground  for  any  public  purpose.  In  individual 
instances  such  examination  may  become  the  duty  of  the  phy- 
sician, when  suspicion  is  aroused  as  to  a  relation  between 
existing  disease  and  soil  or  air.  Of  much  greater  signifi- 
cance in  the  development  of  the  infectious  diseases  is  water, 
concerning  whose  essential  participation  in  the  causation 
of  epidemics  reference  has  been  made  (pp.  170,  190). 
Bacteriologic  examination  of  water  often  devolves  upon-  the 
physician. 

SOIL. 

Method  of  Investigation. — By  means  of  a  sterilized 
platinum  spoon  of  known  capacity  (about  -^^  cu.  cm.)  a 
specimen  of  the  earth  to  be  examined  is  taken  up,  and  with 
this  Esmarch  gelatin  roll-tubes  are  made,  in  which  subse- 
quently the  number  of  colonies  that  have  developed  are 
counted  and  their  nature  is  determined  with  the  aid  of  a 
microscope.  Ordinary  plates  may,  likewise,  be  prepared. 
In  order  that  a  portion  of  the  fragments  of  earth  shall  not 
remain  behind  in  the  test-tube  when  poured  out,  and  the 
result  of  the  estimation  be,  thereby,  rendered  quite  uncer- 
tain, especial  care  must  be  taken  to  secure  uniform  distribu- 

389 


390 


CLINICAL   BACTERIOLOGY. 


tion  of  the  sample.  To  this  end  mortar  and  pestle  are 
sterilized,  and  one  gram  of  earth  diluted  with  ten  times  the 
amount  of  sterile  0.6  per  cent,  solution  of  sodium  chlorid  is 
rubbed  up.  By  means  of  a  graduated  platinum  spiral  a 
definite  amount  is  removed,  and  with  this  gelatin-tubes 
(with  three  or  four  dilutions)  are  made  m  the  usual  man- 
ner. 

In  order  to  obtain  deeper  layers  of  earth  a  special  boring 

instrument  (Fig.  84),  devised  by  C.  Frankel,  is  employed ; 

this  can  be  opened   at  the   desired  depth   by  a    rotatory 

movement,  and,  when  it  is  filled,  is  again 

closed  and  removed. 

By  these  methods,  however,  informa- 
tion is  gained  only  with  regard  to  the 
aerobic  bacteria  present  in  the  earth. 
As,  however,  anaerobic  microorganisms 
occur  in  the  soil,  especially  in  manured 
garden-earth,  in  not  inconsiderable  num- 
ber, it  is  advisable  in  making  bacterio- 
logic  examinations  of  earth  always  to 
prepare  also  anaerobic  plates.  If  it  be 
desired  to  isolate  a  certain  bacterium 
from  the  earth,  animal  experimentation 
is  resorted  to  if  the  organism  is  patho- 
genic. If  the  bacterium  sought  pos- 
sesses resistant  permanent  forms,  the 
sample  of  earth  may  be  distributed  in 
water  and  heated  for  a  considerable 
length  of  time  to  between  80°  C.  (176° 
F.)  and  90°  C.  (194°  F.),  and  then  plates 
are  made. 

The  Bacteria  of  the  Soil. — The  superficial  layers  of 
earth,  even  when  uncultivated,  contain  large  numbers  of 
bacteria — about  100,000  to  the  cubic  centimeter,  and  more. 
The  further  progress  is  made  in  depth  the  smaller  the 
number  of  bacteria  present  in  earth,  and  unless  coarse 
gravel  be  present,  germs  will  no  longer  be  found  at  a  depth 
of  between  ^  and  i  j^  meters,  thus  at  the  ground-water 
area.  Porous  soil  filters  the  air,  as  well  as  liquids,  in  a 
perfectly  reliable  manner.  This  is  naturally  not  the  case 
in  regions  where  fissures  and  breaks  of  any  origin  are 
present. 

Bacilli  especially  are  found  in  earth.     Of  innocuous  mi- 


Fig:.  84.  —  Frankel's 
instrument  for  obtain- 
ing earth  from  various 
depths  for  bacteriologic 
study. 


SOIL.  391 

crobes,  the  hay -bacillus,  the  potato-bacillus,  and  the  root- 
bacillus  (p.  404)  are  cultivated  from  earth  with  great  fre- 
quency. Upon  the  bacteria  of  earth  devolves  the  function  of 
disintegrating  dead  organic  substances,  and  converting  them 
into  materials  from  which  new  organic  substances  (plants) 
are  formed.  The  carbon  of  organic  substances  is  trans- 
formed into  carbon  dioxid  ;  the  nitrogen,  into  ammonia  ;  and 
the  hydrogen,  into  water.  Certain  bacteria  of  the  earth  oxi- 
dize the  ammonia  into  nitrites  (ferments  nitreux,  nitroso-bac- 
teria)  ;  others,  the  nitrites  into  nitrates  (ferments  nitriques, 
nitro-bacteria) ;  the  entire  procedure  of  the  transformation 
of  organic  nitrogen  into  nitric  acid  is  designated  nitrification. 
This  transformation-process,  which  is  brought  about  by  the 
bacterial  flora  of  the  earth,  is  absolutely  indispensable  to 
the  vegetable  world.  If  plants  are  placed  in  soil  that  con- 
tains all  necessary  nutritive  elements,  but  has  been  sterilized 
artificially — that  is,  rendered  bacteria-free — they  will  de- 
velop but  incompletely,  and  soon  begin  to  die  (Duclaux). 
It  must  be  pointed  out  that  these  nitrifying  bacteria  develop 
also  in  cultures  that  contain  no  trace  of  organic  carbon- 
compounds,  and  that  they,  therefore,  obtain  their  carbon- 
requirement  directly  from  the  carbon  dioxid,  without  the 
aid  of  chlorophyl  and  light. 

In  the  superficial  layers  of  the  earth,  in  addition  to  the 
bacilli,  vcvaxvy  permanent  spores  are  present,  which  in  part 
possess  extraordinary  resistance,  and  withstand  for  four  or 
five  hours  the  action  of  live  steam.  Favorable  conditions 
for  spore -formation  must,  therefore,  be  present  in  the  super- 
ficial layers  of  the  soil.  Perhaps  the  circumstance  is  of 
significance  that  the  individual  particles  of  earth  are  sur- 
rounded by  a  capillary  zone  of  fluid,  and  are  thus  protected 
from  drying.  It  has  been  shown  that  anthrax-bacilli  in 
cultures  mixed  with  porous  particles  of  earth  undergo- 
sporulation  much  more  quickly  and  actively  than  otherwise.. 
Anthrax-spores  retain  their  infectivity  for  years  in  earth  in: 
which  dissected  animal  carcases  have  been  buried.  Of 
pathogenic  bacteria,  cultivated  and  manured  earth  fre- 
quently contains,  besides,  the  bacillus  of  tetanus  and  that  of 
malignant  edema. 

The  malarial  parasites,  likewise,  must  reside  in  the  earth 
in  certain  localities  ;  at  least,  numerous  clinical  facts  support 
such  a  view.  Their  demonstration  therein  has,  however, 
as  yet  not  been   successful.     Other  infectious  agents  have 


392  CLINICAL  BACTERIOLOGY. 

up  to  the  present,  been  found  in  the  earth  only  when  but  a 
short  time  has  elapsed  after  the  bacteria  in  question  have 
been  introduced  at  the  given  point  with  the  disease-pro- 
ducts (cholera  or  typhoid  dejections,  etc.).  Typhoid-bacilli 
deposited  at  a  depth  of  J^  meter  retain  their  capability  of 
development,  under  favorable  conditions,  for  five  and  a  half 
months,  and  perhaps  still  longer. 

On  the  whole,  it  may  be  stated  that  pathogenic  germs 
may  multiply  upon  and  within  the  surface  of  the  earth  if 
the  atmospheric  temperature  be  high  ;  but  that,  as  a  rule, 
they  are  suppressed  by  the  competition  of  saprophytes. 
In  the  deeper  layer  the  conditions  for  their  propagation 
are  much  less  favorable. 

The  burial  of  the  bodies  of  individuals  that  have  died 
from  infectious  diseases  is  scarcely  capable  of  giving  rise  to 
infection.  The  accompanying  bacteria  are  overrun  by  the 
saprophytes,  and  even  if  a  small  number — ^^as,  for  instance,  of 
tubercle-bacilli — may  persist  for  months,  perhaps  for  years, 
the  opportunity  is,  on  the  whole,  seldom  afforded  for  their 
being  carried  from  a  depth  to  the  surface  of  the  earth  or 
into  the  subsoil  water. 


AIR. 

Method  of  Examination. — /.  Procedure  of  Hesse. — A 
glass  tube  70  cm.  long  and  3.5  cm.  in  diameter  is  sterilized 
and  coated  with  gelatin,  which,  in  the  same  way  as  in 
Esmarch's  tubes,  is  distributed  uniformly  upon  the  inner 
surface  by  rotation  in  cool  water.  By  means  of  an  aspirator 
air  is  then  drawn 'through  this  tube  at  the  rate  of  a  liter  in 
from  two  to  four  minutes.  At  this  slow  rate  of  speed  the 
germs  of  the  air  are  deposited  upon  the  gelatin,  in  which 
they  subsequently  develop  into  colonies.  This  method 
permits  of  examination  of  only  relatively  small  quantities 
of  air  (from  10  to  20  liters). 

2.  Procedure  of  Petri. — A  sand-filter,  3  cm.  thick,  sup- 
ported upon  two  wire  nets,  is  fastened  in  a  short,  glass  tube, 
with  a  diameter  of  from  1.5  to  2  cm.  ;  the  whole  is  steril- 
ized, and  air  is  permitted  to  stream  through  in  a  rapid  cur- 
rent. The  sand,  which  should  have  a  grain  of  from  ^  to 
^  mm.  in  size,  filters  with  certainty  all  of  the  germs  con- 
tained in  the  air.  After  from  50  to  100  liters  of  air  have 
been  drawn  through  the  apparatus,  the  entire  filter  is  intro- 


AIR. 


393 


duced  into  liquefied  gelatin  or  agar,  and  plates  are  made. 
The  amount  of  air  is  measured  by  means  of  a  gas-meter. 

3.  If  a  precise  quantitative  result  is  not  desired,  gelatin- 
plates  may  simply  be  exposed  to  the  air  for  a  certain  period 
of  time,  and  the  germs  deposited  may  be  permitted  to  de- 
velop into  colonies. 

Bacteria  of  the  Air. — One  cubic  meter  of  air  contains, 
on  the  average,  from  500  to  1000  germs,  including  from 
100  to  200  bacteria.  Molds  constitute  by  far  the  majority 
of  the  germs  ;  next  in  frequency  are  yeast-fungi,  and  last, 


Fig.  85.— Hesse's  apparatus  for  collecting 
bacteria  from  the  air. 


Fig.  86.— Petri's  sand-filter  for 
air-examination. 


bacteria,  which  are  usually  represented  only  by  micrococci 
and  sarcinae. 

The  number  of  germs  present  in  the  air  is  in  marked 
degree  dependent  upon  local  and  temporal  variations.  In 
inhabited  localities,  where  dust  is  being  constantly  created 
and  blown  about,  the  air  contains  a  larger  number  of  micro- 
organisms than  in  deserts.  On  uninhabited  mountains  the 
air  is  almost  entirely  free  from  germs,  as  it  is  also  at  sea. 
At  a  short  distance  from  land  sea-air  contains  germs  carried 
thither  from  the  land  by  currents  of  air.     After  a   heavy 


394  CLINICAL  BACTERIOLOGY. 

rainfall  and  during  the  winter  the  number  of  germs  in  the 
air  is  considerably  diminished.  In  the  still  air  of  rooms 
comparatively  few  germs  are  found,  even  in  densely  occu- 
pied quarters — as,  for  instance,  in  hospital-wards.  As 
soon,  however,  as  dust  is  set  in  motion,  the  number  of 
microorganisms  in  the  air  increases  enormously — to  as 
many  as  i6,ooo  in  the  cubic  meter.  The  majority  of  the 
bacteria,  however,  by  reason  of  their  weight,  are  soon 
again,  after  from  half  an  hour  to  an  hour,  deposited  upon 
the  floor  and  the  walls  with  the  dust,  and  the  air  will  then 
contain  almost  only  the  light  mold-spores. 

Sources  of  Aerial  Germs  (Air-infection). — The  pres- 
ence of  spores  of  molds  in  the  air  in  such  large  number  is 
explained  by  the  fact  that  generally  the  fruit-bearers  of  the 
fungous  deposits  project  upward  from  the  mycelium,  and 
the  spores  can,  therefore,  be  readily  conveyed  by  currents 
of  air. 

Bacteria  frequently  find  their  way  into  the  air  with  the 
small  fibers  separated  from  clothing,  handkerchiefs,  linen, 
etc. 

With  regard  to  the  possibility  of  air-infection,  recent  in- 
vestigations by  Fliigge  and  his  pupils  have  yielded  a  wealth 
of  facts  that  are  well  adapted  to  modify  the  views  hitherto 
held.  The  earlier  supposition  that  no  germs  enter  the  air 
from  liquids  or  from  moist  surfaces  is,  according  to  Fliigge, 
valid  only  in  so  far  as  the  surface  of  the  fluid  remains  un- 
moved and  intact.  A  moderate  wind  (a  velocity  of  four 
meters  in  a  second)  suffices,  however,  to  set  free  germ- 
containing  small  drops  from  water-surfaces,  moist  articles, 
and  the  like.  The  conditions  favoring  the  conversion  of 
fluids  into  small  drops  in  the  air  are  often  present,  as  in  the 
open  air,  in  a  high  degree  in  the  neighborhood  of  the 
surging  sea,  of  water-mills,  etc.,  in  lesser  degree  with  every 
current  of  air  that  sets  the  leaves  of  trees  in  motion.  In 
closed  apartments,  according  to  *  Fliigge,  small  drops  that 
enter  the  air  are  set  free  more  frequently  than  is  generally 
believed  in  sprinkling,  in  washing,  in  manipulating  wet 
linen,  and — upon  which  especial  weight  must  be  placed — 
in  speaking,  sneezing,  and  coughing.  These  droplets, 
whether  they  contain  germs  or  not,  are,  as  Fliigge  further 
demonstrated,  carried  away  by  the  slightest  currents  of  air 
for  considerable  distances,  and  not  only  in  a  horizontal,  but 
also  in  a  vertical  direction.      In  order  to  demonstrate  the 


AIR.  395 

dissemination  of  such  droplets  in  speaking,  coughing,  and 
sneezing,  Fliigge  had  the  person  under  observation  intro- 
duce a  suspension  of  prodigiosus  into  the  mouth.  All  the 
observations  yielded  positive  results  :  The  plates  exposed 
at  a  distance  of  several  meters  became  covered  by  char- 
acteristic colonies,  and  they  remained  sterile  only  after  quiet 
speaking  in  a  low  tone. 

With  regard  to  the  separation  of  desiccated  germs,  it  may 
be  said  that  this  may  likewise  occur  frequently.  Fliigge 
found  that  the  movement  of  the  finest  dust  already  begins 
with  a  current  of  air  having  a  velocity  of  one  meter.  In 
the  open  air  the  most  varied  mechanical  influences  (wagon- 
wheels,  pedestrians)  cause  the  separation  of  the  smallest 
particles,  which  are  then  readily  borne  by  the  wind.  In 
rooms  particles  of  dust  and  fibers  are  set  free  by  the  vibra- 
tion of  the  floor,  by  manipulation  of  utensils,  furniture, 
clothing,  etc.  The  smallest  of  these  floating  particles  con- 
taining germs  are,  however,  as  Fliigge  has  shown,  moved 
in  a  horizontal  direction  by  minimal  currents  of  air  of  0.2 
mm.,  and  in  a  vertical  direction  by  currents  of  from  0.3  to 
0.4  mm.  per  second.  In  the  course  of  his  experiments 
Fliigge  succeeded  with  the  prodigiosus  in  demonstrating 
the  distribution  of  dust-germs  in  all  possible  parts  of  a 
closed  room.  Germ-containing  dust  is  not  completely 
removed  from  rough  surfaces  even  by  the  strongest  cur- 
rents of  air.  Airing  of  infected  clothing,  as  frequently 
practised,  does  not,  therefore,  entirely  attain  the  object 
desired. 

Upon  the  basis  of  the  results  of  these  observations 
Fliigge  expresses  the  view  that  in  the  case  of  all  infectious 
diseases  air-infection  may  take  place  through  the  smallest 
droplets  separated  from  fluid  sources  of  infection.  In 
cholera  and  typhoid  fever  this  will  occur  but  rarely — for  in- 
stance, in  the  sprinkling  of  polluted  water,  in  the  washing  of 
linen,  and  the  like.  The  principal  role  in  these  affections 
is  played  by  contact-infection,  the  dissemination  through 
drinking-water,  articles  of  food,  etc.  In  the  infectious  dis- 
eases of  the  nose,  the  pharynx,  and  the  respiratory  organs, 
however,  the  possibility  of  transmission  through  small 
drops  must  be  given  greater  consideration  than  it  has  re- 
ceived in  the  past.  In  diphtheria,  influenza,  whooping- 
cough,  pneumonia,  and  pulmonary  tuberculosis,  droplets 
containing    germs   are    sent   into    the    air    in    coughing, 


396  CLINICAL  BACTERIOLOGY. 

sneezing,  and  loud  talking ;  they  are  set  in  motion  by  min- 
imal currents  of  air,  and  in  a  quiet  room  can  be  demon- 
strated floating  in  the  air  for  as  long  as  five  hours. 

In  cases  of  air-infection  through  dry  particles  of  dust, 
only  the  finest  particles,  capable  of  being  carried  by  any 
movement  of  air,  demand  consideration.  Only  such  diseases 
are  transmitted  naturally  in  this  way  whose  exciting  agents 
are  still  capable  of  surviving  in  the  dry  state.  For  the 
acute  exanthemata,  which  have  always  been  considered  as 
diseases  due  to  volatile  contagia,  such  a  possibility  must  be 
accepted. 

Pulmonary  tuberculosis  has  hitherto  been  generally 
accepted  as  disseminated  through  inhalation  of  ejected 
dried  and  pulverized  sputum  containing  tubercle-bacilli  ; 
Fliigge  opposes  this  view.  Attempts  to  infect  animals 
through  inhalation  of  dried  tuberculous  sputum  have  never 
quite  succeeded  ;  and,  further,  the  tubercle-bacilli  do  not 
appear  at  all  capable  of  being  carried  by  the  finest  particles 
of  dust.  Fliigge  expresses  a  warning  against  going  to  the 
other  extreme  of  attaching  too  great  importance  to  the 
danger  of  infection  through  drops  of  spray  or  even  of  con- 
sidering it  the  only  source  of  infection.  Infection  with 
tuberculosis  is  dependent  upon  a  large  number  of  factors  : 
upon  the  environment  of  the  patient,  upon  the  presence 
of  tubercle-bacilli  in  the  saliva,  which  forms  fine  droplets 
much  more  readily  than  the  viscid  sputum  proper,  and 
upon  other  conditions. 


WATER. 

Method  of  Examination. — The  water  to  be  examined 
is  collected  in  sterilized  Erlenmeyer  flasks,  and  is  investi- 
gated as  speedily  as  possible.  If  the  examination  is  deferred 
for  only  a  few  hours,  the  indifferent  saprophytic  bacteria 
that  reside  in  water  will  have  undergone  multiplication,  and 
the  estimation  of  the  number  of  water-germs  will  not  yield 
reliable  results.  If  the  specimens  of  water  to  be  examined 
are  sent  from  a  distance,  the  water  must  be  forwarded  in 
sterilized  flasks,  provided  with  glass  stoppers,  and  packed 
in  ice.  In  obtaining  water  for  examination  care  should  be 
taken  that  the  material  to  be  examined  has  not  stagnated 
in  the  conduit  from  the  well  or  spring  or  other  source  of 
supply.      For  this  reason  a  certain  amount  of  water  is  first 


WATER.  397 

permitted  to  escape.  Of  the  specimen  obtained,  i,  ^,  and 
i^  cu.  cm.  are  removed  by  means  of  sterilized  pipets,  and 
introduced  into  liquefied  gelatin,  and  plates  are  made. 
Greatly  polluted  water  should  be  diluted  with  sterilized 
water  from  another  source,  and  no  more  than  -^,  yi^,  and 
Y^-Q-Q  CU.  cm.  should  be  employed.  Before  introducing  the 
pipets  the  specimen  is  vigorously  agitated,  as  the  bacteria, 
by  reason  of  their  weight,  quickly  settle  to  the  bottom.  In 
the  process  of  water-examination  Koch's  old  method  of 
making  plates  is  .still  frequently  followed,  because  these 
permit  better  than  the  Petri  dishes  the  distribution  of  the 
gelatin  in  as  uniform  a  layer  as  possible,  \yhen  the  plates 
have  developed,  the  colonies  are  examined  and  counted,  if 
present  in  large  number  by  means  of  a  special  enumerator 
(a  glass  plate  with  etched  squares).  An  estimate  is  made 
of  the  number  of  germs  to  the  cubic  centimeter  of  water, 
and  the  number  as  well  as  the  identity  of  the  varieties 
present  is  determined. 

The  Number  of  Bacteria  Present  in  Water. — Spring- 
water,  as  well  as  subsoil-water,  is  free  from  germs  at  the 
point  where  it  escapes  from  the  earth.  It  has  been  men- 
tioned that  the  earth  at  the  level  of  the  subsoil-water  no 
longer  contains  bacteria.  According  to  the  most  trust- 
worthy investigations,  pure  tap-water  and  spring-water  con- 
tain, on  the  average,  from  2  to  50  bacteria  per  cubic  centi- 
meter ;  pure  pump-water,  from  lOO  or  200  to  500  germs  ; 
unfiltered  water  from  streams  kept  unpolluted,  from  6000  to 
20,000 ;  filtered  river-water,  from  50  to  200 ;  polluted 
wells,  as  many  as  100,000  germs  ;  and  river  tap-water, 
when  the  filtering  apparatus  is  out  of  order,  the  same  num- 
ber. Drain-water  and  greatly  contaminated  streams  contain 
from  2,000,000  to  40,000,000  germs  to  the  cubic  centimeter 
(Fliigge).  In  the  summer  and  after  a  heavy  rainfall  the 
number  of  bacteria  in  water  is  increased. 

The  Bacteria  of  Water.— The  microorganisms  living  in 
water  are  mainly  bacilli.  A  large  number  of  them  liquefy 
gelatin ;  others  generate  offensive  gases,  and  still  others 
beautiful  pigments.  Of  great  interest  are  the  so-called 
typhoid-like  water-bacteria  (p.  402)  and  the  water-vibrios, 
which  in  a  number  of  points  resemble  the  comma-bacilli 
of  Asiatic  cholera  (p.  404). 

Among  pathogenic  bacteria  typhoid-bacilli  and  cholera- 
vibrios  have  been  repeatedly  found  in  water.     The  method 


398  CLINICAL  BACTERIOLOGY. 

for  cultivating  these  bacteria  from  water  has  been  consid- 
ered in  the  discussion  of  typhoid  fever  (p.  178)  and  of  cholera 
(p.  195).  At  this  point  it  may  be  briefly  repeated  that  by 
the  addition  of  one  per  cent,  peptone  and  ^  per  cent, 
sodium  chlorid,  the  water  to  be  examined  itself  constitutes 
a  nutrient  medium,  so  that  in  this  way  large  quan- 
tities of  the  fluid  can  be  employed,  whereas  in  earlier 
investigations  a  fraction  of  a  drop  had  to  suffice,  and,  as  a 
result,  the  pathogenic  germs  were  readily  overlooked. 

The  saprophytic  water-bacteria  multiply  in  water  to  an 
unlimited  degree.  For  the  pathogenic  bacteria,  however, 
while  the  possibility  of  preservation  often  exists,  but  rarely 
is  opportunity  for  proliferation  in  water  afforded.  To  this 
end  there  are  necessary  a  favorable  external  temperature 
(summer)  and,  further,  solid  particles  of  vegetable  or  ani- 
mal origin,  to  which  the  bacteria  adhere,  which  serve  as 
nutrient  medium,  and  which,  at  the  same  time,  afford  the 
bacteria  protection  from  the  competition  of  saprophytes. 
The  exciting  agents  of  typhoid  fever  and  of  cholera  appear 
to  survive  in  ordinary  water  for  days,  and  even  for  weeks. 
Some  observers  even  believe  that  cholera-vibrios  may  at 
first  undergo  multiplication  in  water.  As  a  rule,  however, 
the  pathogenic  germs  are  suppressed  sooner  or  later  in 
water  in  consequence  of  overgrowth  by  the  saprophytes. 

Self-purification  of  Water. — The  microorganisms  pres- 
ent in  water  are  derived  from  the  surface  of  the  earth,  the 
air,  from  waste  water  and  the  sewage  of  cities,  emptying 
into  the  water-courses,  from  cesspools  communicating  with 
imperfectly  constructed  subsoil-wells,  etc.  The  pollution  of 
streams  by  cities  is  a  most  prominent  factor ;  the  Seine  at 
Ivry,  for  instance,  contains  32,500  germs  to  the  cubic  cen- 
timeter ;  below  Paris,  in  Asnieres,  the  number  is  12,800,000. 
Fortunately,  if  renewed  contamination  does  not  take  place, 
streams  purify  themselves  (Pettenkofer).  The  microor- 
ganisms settle  and  are  carried  to  the  bottom,  partly  with 
the  constituents  suspended  in  the  water  and  with  the  in- 
soluble earthy  combinations  that  form  from  calcium  and 
magnesium  bicarbonates  after  escape  of  the  carbon  dioxid. 
Light  also  in  a  high  degree  exerts  an  injurious  influence 
upon  the  microorganisms  present  in  water  down  to  a 
depth  of  about  two  meters.  The  organic  substances  con- 
tained in  water  are  gradually  consumed  by  bacteria  and 
algae. 


WATER.  399 

Sources  and  Mode  of  Water-supply. — In  the  practical 
application  of  bacteriologic  examination  of  water  for  the 
construction  of  conduits,  wells,  etc.,  it  is,  in  the  first  place, 
important  to  determine  whether  the  water  used  contains 
pathogenic  bacteria  or  not.  If  a  given  water  contains 
many  putrefactive  bacteria  (proteus)  or  the  bacterium  coli 
commune,  it  should  be  entirely  excluded  from  use  ;  for 
such  conditions  indicate  with  certainty  that  the  source  of 
supply  is  polluted.  Formerly,  great  weight  was  attached 
to  the  number  of  germs  present.  Some  difficulty  was  en- 
countered in  this  connection  in  the  establishment  of  a  limit, 
beyond  which  water  should  no  longer  be  considered  pure. 
Some  observers  made  this  limit  one  hundred,  others  fifty, 
and  still  others  five  hundred.  The  absolute  number  of 
germs  contained  in  a  given  water  depends,  however,  upon 
such  varied  factors  that  an  opinion  based  solely  upon  the 
number  of  colonies  formed  is  not  to  be  depended  upon.  It 
was  then  proposed  to  attach  the  greatest  importance  to  the 
number  of  different  varieties  present.  A  large  number  of 
different  species,  it  was  thought,  would,  to  a  certain  degree, 
be  an  indication  of  the  greater  probability  of  the  occur- 
rence of  contamination  of  the  water.  This  belief  is  not 
without  justification,  but  too  much  significance  must  not  be 
attached  to  the  number  of  different  varieties  of  bacteria  in 
water.  The  fact  can  not  be  evaded  that  bacteriology  has 
thus  far  not  attained  the  importance  in  the  hygienic  deter- 
mination of  the  usefulness  of  water  that  was  originally 
attributed  to  it. 

As  a  central  source  of  water-supply  for  large  communi- 
ties bacteria-free  spring-water  or  subsoil-water  is  most  to  be 
recommended,  when  this  is  obtainable  in  at  all  sufficient  quan- 
tities. In  the  selection  of  a  source  of  supply  care  should 
be  taken  to  avoid  undue  proximity  to  communities  or 
manured  land.  If  spring-water  or  subsoil-water  is  not 
available,  there  is  no  alternative  but  to  employ  river-water 
or  lake-water,  which  naturally  must  be  obtained  from  a 
point  above  the  city  to  be  supplied.  Such  water  is,  as  has 
been  pointed  out,  exposed  to  numerous  sources  of  contami- 
nation, and  must,  therefore,  unconditionally  be  subjected  to 
some  process  o^ filtration  before  being  used.  This  maybe 
effected  by  means  of  sandfilti'ation,  which  is  carried  out  in 
large,  cemented,  covered  reservoirs.  The  utility  of  sand-fil- 
ters has  been  studied  in  recent  years  with  especial  care  by 


400  CLINICAL  BACTERIOLOGY. 

C.  Frankel  and  Pief  ke.  At  the  bottom  of  a  filtering  basin 
large  cobblestones  should  be  placed  to  a  height  of  305  mm., 
upon  these  a  layer  of  small  cobblestones,  102  mm.  high, 
then  76  mm.  of  coarse  gravel,  127  mm.  gravel  of  medium 
size,  upon  this  51  mm.  of  coarse  sand,  and,  finally,  559  mm. 
of  fine  sand.  Filtering  properties  reside  solely  in  the  layer 
of  sand.  Before  the  process  of  filtration  is  begun  the 
reservoir  must  be  permitted  to  remain  filled  with  water  for 
twenty-four  hours.  As  a  result,  there  will  form  a  coating 
of  sediment,  and  a  slimy  covering  for  the  pores  of  the 
filter,  which  constitute  the  essential  factors  in  the  purifica- 
tion of  the  water.  The  rate  of  filtration  should  not  ex- 
ceed 100  mm.  an  hour.  The  form  of  filter  described  does 
not  yield  perfectly  sterile  water,  but  this  will  be  found  to 
contain  between  50  and  200  germs  in  the  cubic  centimeter, 
which  it  must  be  noted  are  derived  in  largest  part  from  the 
lower  layers  of  the  filter.  The  mechanism  can  be  disturbed 
by  tears  in  the  cover  of  the  filter,  when  the  filtration- 
pressure,  which  will  increase  with  increasing  sliminess  of 
the  filter,  becomes  too  great.  The  filter  must,  therefore, 
be  cleansed  from  time  to  time.  The  working  of  the  filter 
altogether  demands  unremitting  attention.  The  water  of 
every  filter  must  be  examined  bacteriologically  every  day, 
and  as  soon  as  more  than  100  bacteria  are  contained  in  the 
cubic  centimeter,  use  of  the  water  should  not  be  permitted, 
and  the  filter  should  be  renewed. 

Of  house -jilt  ei's  for  the  purification  of  water  for  domestic 
use  numerous  varieties  have  been  recommended.  Of  all 
not  one  properly  serves  its  purpose,  as  they,  are  soon  sat- 
urated with  bacteria.  For  domestic  purposes  suspected 
water  is  rendered  harmless  in  the  simplest  manner  by  boil- 
ing for  five  minutes  (after  the  vapor  of  steam  has  arisen). 
Of  wells  the  best  as  a  source  of  water-supply  are  of  the 
tubular  variety,  in  which  an  iron  tube  leads  to  the  subsoil- 
water  area,  and  thus  furnishes  germ-free  water. 

Ice.- — Natural  ice,  which  is  obtained  in  winter  from 
streams  and  ponds,  contains  numerous  bacteria — on  an 
average  2000  to  the  cubic  centimeter  in  ice-water,  with  a 
minimum  of  50  and  a  maximum  of  25,000  germs.  Quite 
a  number  of  bacteria,  among  them  the  cholera-vibrios,  offer 
considerable  resistance  to  freezing.  Some  even  are  capable 
of  multiplication  at  this  temperature  (p.  22).  Artificial  ice 
prepared  from   distilled  water  contains  from  none   to    10 


BACILLI   IN  WATER.  401 

germs  in  the  cubic  centimeter  of  ice- water.  Distilled  water 
itself,  after  standing  for  some  time,  contains  many  water- 
bacteria,  but  these  are  among  those  that  do  not  bear 
freezing. 

Artificial  carbonated  waters  are  often  rich  in  bacteria, 
even  after  standing  for  months,  and  it  has  been  shown  ex- 
perimentally that  typhoid-bacilli,  for  instance,  may  survive 
for  from  days  to  weeks  in  such  water.  Care,  therefore, 
should  be  taken  to  prepare  artificial  waters  only  from  pure 
drinking-water  or  from  distilled  water. 


THE  BACTERIA  PRINCIPALLY  FOUND  IN  SOIL, 
AIR,  AND  WATER* 

L  BACILLL 

I.  NOT  LIQUEFYING  GELATIN. 

(a)  Chromogenic. 

1.  Bacillus  Aurantiacus . — This  is  a  short,  plump  rod,  with 
slight  spontaneous  movement.  Upon  plates  it  appears  in  the 
form  of  an  orange-colored,  knob-like  deposit.  In  gelatin  stab- 
cultures  it  exhibits  a  glistening,  orange-colored  growth.  Its 
appearance  in  bouillon  is  characteristic.  The  fluid  itself  remains 
clear,  while  upon  the  surface  a  membrane  forms,  presenting  a 
small  number  of  orange-colored  spots,  and  at  the  bottom  a 
somewhat  lighter  sediment  collects. 

2.  Bacillus  Constrictus. — This  organism  derives  its  name 
from  the  peculiar  appearance  it  presents  when  stained  by  the 
method  of  Zimmermann.  The  rods  exhibit  a  slight  constriction 
between  the  individual  segments,  which  are  united  into  short 
chains,  and  they  resemble  biscuits  in  shape.  In  plates  the  colo- 
nies present  the  appearance  of  granular  discs,  with  eroded  mar- 
gins. The  color  is  between  yellowish-gray  and  light  sulphur- 
yellow. 

J.  Bacillus  Fluorescens  Nonliquefaciens. — This  is  a  delicate, 
short,  actively  motile  rod.  Upon  gelatin  the  colonies  present 
a  peculiar  mother-of-pearl  luster,  which  also  exhibits  fluores- 
cence. Upon  agar-agar  the  growth  acquires  a  greenish  tint.  A 
subvariety  has  been  described  as  bacillus  fiuorescens  nonliquefa- 
ciens immobilis,  which  is  distinguished  by  the  absence  of  motility 
and  of  flagella. 

4.  Bacillus  Fuscus. — This  is  a  medium-sized  rod,  sometimes 
curved,  which  takes  its  name  from  the  dark-brown  pigment  to 
26 


402  CLINICAL  BACTERIOLOGY. 

which  it  gives  rise  in  all  nutrient  media.  In  gelatin  stab- 
cultures  a  nail-shaped  growth  develops  at  first,  the  head  subse- 
quently extending. 

5.  Bacillus  Rubefaciens. — This  is  a  fine  rod,  consisting  of 
two  or  more  segments.  Gelatin-cultures  exhibit  a  pale  rose-red 
color.  Upon  potatoes  the  substratum  appears  of  a  rose-red 
color,  while  the  colony  itself  is  between  yellowish -gray  and 
brownish-red. 

6.  Bacillus  Subflavus. — Cultures  of  this  organism  give  rise 
to  a  pale-red  pigment;  upon  plates  they  exhibit  a  mother-of- 
pearl  luster.  The  pigmentation  is  most  distinct  in  agar-agar 
cultures.  Several  bacilli  often  lie  attached  to  one  another. 
The  individual  organism  is  from  two  to  four  times  as  long  as 
it  is  thick. 

7.  Bacillus  Brunneus. — The  colonies  of  this  small,  nonmotile 
bacterium  are  characterized  by  diffusing  a  brownish  pigment  into 
the  surrounding  culture-medium. 

(b)  Nonchromogenic. 

8.  Ty phoid- like ,  Bacilli  (^Weichselbaum) . — Under  this  desig- 
nation is  included  a  group  of  motile  bacilli  that  resemble 
the  bacillus  of  Eberth-Gaffky  and  the  bacterium  coli  commune 
in  both  morphologic  and  cultural  properties.  On  plates  the 
colonies  present  easily  the  appearances  of  those  of  the  typhoid- 
bacillus  and  the  colon-bacillus  (pp.  167,  120).  Upon  potatoes 
a  deposit  forms,  at  times  brownish,  at  other  times  yellowish,  at 
still  other  times  scarcely  visible.  Coagulation  is  induced  in 
milk.  Some  varieties  cause  fermentation  of  grape-sugar,  while 
others  do  not.  The  nitroso-indol  reaction  is  positive  with  some, 
and  negative  with  others.  These  bacteria  are  free  from  all 
pathogenic  activity  in  experiments  on  animals.  That  this  group 
is  constituted  of  a  series  of  bacteria  differing  among  themselves 
is  demonstrated  by  the  fact  that  any  one  variety  is  incapable  of 
conferring  immunity  to  any  other.  The  attempt,  also,  to  cause 
agglutination  in  the  cultures  of  one  variety  with  the  blood- 
serum  of  animals  that  have  been  immunized  to  another  variety 
has  invariably  failed. 


2.  LIQUEFYING  GELATIN. 

(a)  Chromogenic. 

9.  Bacillus  Arborescens. — This  is  a  slender  bacillus  that  fre- 
quently forms  wavy  filaments.  It  is  incapable  of  spontaneous 
movement,  and  it  is  characterized  by  branch-like  ramifications  in 
gelatin  plate-cultures,  and  by  iridescence  of  its  colonies.  It 
gives  rise  to  a  yellowish  or  yellowish-red  pigment,  especially 
upon  potatoes. 

10.  Bacillus   Fluorescens    Liquefaciens . — This    is    a    motile 


BACILLI   IN  WATER.  403 

organism  that  closely  resembles  the  bacillus  pyocyaneus.  It 
liquefies  gelatin  rapidly,  with  the  formation  of  a  greenish-yellow 
pigment,  which  is  vividly  fluorescent.  Typical  cultures  form 
upon  glycerin-agar,  which  acquirer  from  an  olive-green  to  a  dark 
olive-brown  color. 

11.  Bacillus  Rubidus. — This  is  a  medium-sized  rod,  actively 
motile,  and  arranged  in  threads  of  considerable  length.  It 
generates  a  brownish-red  pigment,  both  in  gelatin  and  in  agar, 
and  also  in  potato-cultures.  Other  than  pigment-formation  it 
presents  scarcely  anything  else  characteristic. 

12.  Bacillus  Violaceus. — This  is  a  small,  slender,  actively 
motile  rod,  that  upon  agar  forms  central  spores.  In  its  growth 
upon  gelatin-plates  a  bluish-violet  bacterial  mass  appears  in  the 
liquefied  culture-medium.  Upon  agar-agar  and  potatoes  the 
formation  of  pigment  is  intense,  and  the  color  is  dark  violet, 
almost  black. 

I  J.  Bacillus  Viscosus. — This  organism  closely  resembles  the 
bacillus  fluorescens  liquefaciens,  from  which,  however,  it  is 
differentiated  by  the  formation  of  a  chocolate-colored  coating. 

14.  Bacillus  lanthinus. — This  is  a  motile  bacillus  of  medium 
size.  Its  appearance  in  growth  upon  gelatin-plates  is  usually 
compared  with  the  appearance  of  a  drop  of  ink  that  has  fallen 
upon  them.     It  forms  a  violet  pigment  in  all  nutrient  media. 

i^.  Bacillus  Helvolus. — This  appears  in  the  form  of  motile 
rods  of  varying  length,  which  are  oftea  united  into  short  threads. 
These  generate  from  a  yellowish  to  a  sulphur-yellow  pigment. 
Upon  plates  the  colonies  appear  as  circular,  bright-yellow  discs 
that  lie  in  a  funnel  of  liquefaction.  Upon  agar  an  abundant 
deposit  of  intensely  yellow  hue  forms. 

16.  Bacillus  Prodigiosus. — This  is  a  small  rod  (formerly 
designated  micrococcus  prodigiosus  or  monas  prodigiosa),  often 
collected  in  small  chains,  of  slight  motility,  occurring  not  rarely 
in  the  air,  less  commonly  in  water,  rather  frequently  upon  amy- 
laceous nutrient  media  (bread,  potatoes),  upon  meat,  and  in 
milk.  It  grows  upon  all  nutrient  media,  with  the  development 
of  a  bright-red  pigment ;  this  is  most  intense  on  potatoes,  which 
present  a  blood-red  coating.  Upon  gelatin-plates  rather  deep, 
small,  white  dots  appear,  and  .  also  superficial,  roundish,  red 
colonies  with  an  irregular  border.  Gelatin  is  energetically 
liquefied.  Upon  agar-agar  a  coating  of  moderately  dark-red 
color  appears,  while  the  nutrient  medium  itself  is  not  discolored. 
On  cultivation  at  a  temperature  of  37°  C.  (98.6°  F. )  the  pro- 
digiosus loses  its  red  color  in  the  course  of  several  generations.  In 
cultures,  especially  upon  potato,  in  addition  to  the  red  pigment, 
trimethylamin  forms  (odor  of  pickled  herring).  Coagulation 
is  induced  in  milk.  Saccharine  nutrient  media  undergo  fermen- 
tation.    The  prodigiosus  will  thrive  also  in  the  absence  of  oxy- 


404  CLINICAL   BACTERIOLOGY. 

gen,  but  then  without  the  development  of  red  pigment.  It  is 
somewhat  pathogenic,  inoculated  animals  dying  with  toxic 
symptoms  after  introduction  of  large  amounts.  The  name  pro- 
digiosus  is  derived  from  the  fact  that  the  bloody  appearance  of 
the  so-called  miraculous  holy  wafers  is  attributed  to  infection 
with  this  microorganism. 

17.  Among  other  pigment-producing  bacteria  characterized 
only  by  the  color  of  their  cultures  may  be  mentioned  the 
bacillus  ruber  balticus,  bacillus  ruber  aquatilis,  bacillus  coeruleuSy 
bacillus  pavoninus,  bacillus  amethystinus. 

(b)  Nonchromogenic. 

18.  Bacillus  Liquefaciens . — This  is  one  of  the  most  wide- 
spread of  the  water-bacilli.  It  is  an  actively  motile  rod,  often 
joined  in  short  chains  of  four  or  more  segments.  It  rapidly 
liquefies  gelatin.  In  plates  it  assumes  the  form  of  a  dish,  upon 
the  base  of  which  lies  a  gray,  bacterial  mass.  In  stab-cultures 
the  growth  assumes  the  form  of  a  stocking,  with  a  dilated  upper 
portion.  The  odor  of  the  culture  is  highly  disagreeable.  The 
bacillus  exhibits  facultative  anaerobiosis.  In  nutrient  media 
containing  nitrates  it  generates  nitrous  acid. 

The  following  likewise  are  included  among  water-bacteria : 

zp.  Bacillus  Liquidis. — This  is  a  short,  plump,  slightly  motile 
bacillus,  which  also  rapidly  liquefies  gelatin.  In  tubes  the 
gray,  liquefied  gelatin  becomes  covered  with  a  thin  membrane, 
which  sinks  to  the  bottom  on  agitation. 

20.  Bacillus  A quatilis. — This  is  a  slender  rod,  with  spontane- 
ous motility.  It  liquefies  gelatin  slowly,  and  according  to  some 
observers  not  at  all.  It  grows  in  gelatin  upon  the  surface  in  the 
form  of  small,  yellowish  colonies,  and  upon  potatoes  with  a 
scanty  yellow  deposit. 

In  the  earth  and  in  certain  articles  of  food  the  following 
bacteria  are  always  to  be  found,  which  are  characterized  by 
especial  resistance  of  their  spores : 

2j.  Root-bacillus.  — This  is  a  large,  thick  bacillus,  with  rounded 
extremities,  and  possessing  slight  motility.  It  develops  central 
spores,  and  growth  takes  place  only  in  the  presence  of  oxygen. 
The  whitish-gray  colonies  that  form  consist  of  a  network  of  fine, 
interlacing  threads.  They  liquefy  gelatin.  In  stab-cultures 
also  filaments  and  processes  form,  and  an  appearance  results  re- 
sembling an  inverted  fir-tree.  Upon  agar-agar  a  network  forms 
suggesting  the  ramifications  of  the  roots  of  a  tree. 

22.  Bacillus  Subtilis  {^Hay -bacillus'). — This  is  a  large,  deli- 
cate bacillus  that  often  develops  into  long,  straight  threads. 
The  bacillus  subtilis  is  a  strictly  aerobic  organism,  and  it  quickly 
liquefies  gelatin.  The  temperature-optimum  is  30°  C.  (86°  F.); 
the  temperature-minimum,  10°  C.  (50°  F.);  the  temperature- 
maximum,  45°  C.  (113°  F.).     Upon  plates  the  bright,  grayish- 


MICROCOCCI   IN  WATER.  405 

white  colony  appears  surrounded  by  a  sparkling  crown.  Upon 
agar-agar  the  growth  is  peculiar,  a  stiff,  wrinkled,  readily 
detached  deposit  forming.  The  hay-bacillus  forms  central 
spores,  which  are  somewhat  thicker,  but  considerably  shorter 
than  the  mother-cells.  It  is  found  in  the  air,  water,  dust, 
feces,  hay,  etc.  In  order  to  obtain  the  organism  in  pure  culture 
hay  is.  cut  up  into  small  pieces,  which  are  covered  with  water  in 
an  Erlenmeyer  flask,  and  exposed  to  a  boiling  temperature  for 
fifteen  minutes.  In  this  way  all  of  the  germs  are  destroyed, 
with  the  exception  of  the  resistant  spores  of  the  hay-bacillus. 
These  then  grow,  and  after  two  or  three  days  they  form  a  super- 
ficial membrane  upon  the  hay-infusion. 

2j.  Potato-bacillus  (^Bacillus  Mesentericus). — Three  varieties 
of  this  microorganism  are  distinguished  :  bacillus  mesentericus 
vulgatus,  fuscus,  and  ruber.  The  last,  especially,  which  im- 
parts a  rose  tint  to  potatoes  upon  which  it  grows,  possesses  per- 
manent forms  of  extraordinary  resistance,  which  withstand 
boiling  for  from  five  to  six  hours.  In  proportion  to  the  cell  the 
spore  is  quite  large.  The  cultural  peculiarities  of  the  organism 
resemble  those  of  the  hay-bacillus.  Upon  potatoes  the  bacillus 
gives  rise  to  a  wrinkled  coating.  Milk  is  coagulated  and  pep- 
tonized. 

24.  Bacillus  Spinosus. — This  is  a  strictly  anaerobic,  motile 
rod.  Its  colonies  in  gelatin  form  iridescent  globules,  with  thorn- 
like processes.  Gelatin  is  liquefied,  with  the  formation  of  gas. 
Stab-cultures,  before  liquefaction  takes  place,  present  the  appear- 
ance of  a  prickly  caterpillar  (Liideritz).  The  bacillus  spinosus 
grows  both  at  room-temperature  and  at  the  temperature  of  the 
body.  It  forms  central  spores,  the  rod  becoming  at  the  same 
time  spindle-shaped  (Clostridium).  The  bacillus  is  usually 
found  in  garden -earth. 


n,  MICROCOCCL 

I.  NOT  LIQUEFYING  GELATIN. 

(a)  Chromogenic. 

25.  Micrococcus  Aurantiacus. — This  is  a  round  or  oval  coccus, 
arranged  in  groups.  The  cultures  are  yellow,  slimy,  knob- 
shaped,  and  they  do  not  extend  greatly  in  width. 

26.  Micrococcus  Versicolor. — This  is  a  small  coccus,  arranged 
in  groups  or  in  pairs.  It  occurs  with  extraordinary  frequency 
in  the  air.  The  colonies  are  irregular  in  shape,  with  a  yellowish- 
green  color.  They  exhibit,  especially  upon  gelatin,  a  mother- 
of-pearl  iridescence,  and  they  cause  fermentation  in  nutrient 
media  containing  glucose. 


406  CLINICAL  BACTERIOLOGY. 

(b)  Nonchromogenic. 

27.  Micrococcus  Candicans. — This  is  a  round  coccus  of  mod- 
erate size.  Its  most  distinctive  feature  is  its  growth  in  gelatin 
stab-cultures,  in  which  a  nail-shaped  growth  appears,  with  a 
porcelain- white,  glistening  head. 

28.  Micrococcus  Concentricus. — This  is  characterized  by  the 
concentric  extension  of  its  colonies  upon  gelatin-plates  and  in 
stab-culture.  The  colonies  present  from  a  whitish-gray  to  a 
bluish-gray  color,  and  are  superficially  serrated.  The  cocci 
themselves  are  small  and  are  arranged  like  grapes. 

2p.  Micrococcus  Rosettaceus. — This  is  a  coccus  of  moderate 
size.  Its  growth  is  mainly  superficial.  Roset-like  deposits 
form,  with  irregular  margins.  The  colonies  are  grayish-white 
in  color,  but  darker  in  the  center,  up  to  brown. 

30.  Micrococcus  Aquatilis. — The  colonies  are  round,  and 
possess  a  mother-of-pearl  luster.  The  margins  appear  serrated  ; 
the  color  is  light  gray.  Viewed  with  low  powers  of  the  micro- 
scope, the  colony  appears  in  the  form  of  a  berry. 


2.  LIQUEFYING  GELATIN. 

(a)  Chromogenic. 

31.  Micrococcus  Cremoides. — This  is  a  small  coccus,  arranged 
in  groups,  and  giving  rise  to  a  cream- colored  pigment.  At  first, 
the  colonies  upon  gelatin  are  from  yellowish- white  to  brownish- 
gray,  granular,  circular ;  while  later  the  discs  appear  eroded, 
and  they  lie  in  a  liquefied  excavation. 

32.  Sarcina  Lutea  {Yellow  Sarcind). — This  coccus,  strictly 
aerobic,  is  arranged  in  so-called  balls  of  twine.     Upon  gelatin- 
plates  it  forms  rounded,  slightly  granular, 

S^^     ^%  yellow  colonies.     In   stab-cultures  marked 

aD0#»  fiSft        superficial   growth   takes   place.     The  cul- 


••''^A  ^^  ffl»      tures    generate    a    citron -yellow   pigment. 
«flvft^A  «^  ffl^      Liquefaction    occurs  quite    late ;    then  the 


So^^    ^^  £S     clear,  liquefied  gelatin  overlies  the  citron- 
yellow  precipitate.     In  addition  to  the  yel- 
Fig.  87.-Sar_dn^^;  X  600    j^^^   garciua  there  are  also  white,  orange, 
and  red  sarcinse,  which   are   distinguished 
from    that   described    only   by   the  difference  in  color.     The 
varieties  of  sarcinae  are  present  in  the  air. 

jj.  Micrococcus  Agilis    {AH  Cohen). — This  actively  motile 
organism  (flagella),  cultivated  from  drinking-water,  grows  upon 
all  nutrient  media,  with   the  formation  of  a  rose-red  pigment. 
It  slowly  liquefies  gelatin, 
(b)  Nonchromogenic. 

J4.  Micrococcus  Radiatus. — This  is  a  small  coccus,  without 
typical  arrangement.     Upon  plates  it  forms  colonies  surrounded 


VIBRIOS  IN  WATER.  407 

by  a  sparkling  crown.     In  stab-cultures  the  colonies,  likewise, 
display  horizontal  radiation.     Gelatin  is  slowly  liquefied. 


m.  VIBRIOS. 

Since  the  great  epidemic  of  cholera  at  Hamburg  in  the  year 
1892,  a  large  number  of  vibrios  more  or  less  closely  resembling 
that  of  cholera  have  been  described,  which  have  been  cultivated 
in  part  from  river-water,  in  part  from  other  sources,  and  of 
which  the  most  important  will  be  mentioned. 

jj'.  Vibrio  Aquatilis  Gilnther. — This  organism  is  scarcely  to' 
be  confounded  with  the  comma-bacillus,  on  account  of  its  cir- 
cular, finely  granular  colonies,  with  smooth  borders,  even  inde- 


Fig.  88. — Spirillum  aquatilis,  from  an  agar-agar  culture ;  X  1000  (Itzerott  and 
Niemann). 

pendently  of  the  absence  of  the  cholera-red  reaction.  It  at 
first  grew  feebly  in  liquid  nutrient  media,  but  as  a  result  of  re- 
peated inoculation  it  acquired,  in  consequence  of  adaptation  to 
such  media,  the  property  of  growing  upon  bouillon  and  peptone- 
water. 

36.  Vibrio  Berolinensis. — This  organism,  which  was  cultivated 
by  Neisser  (1893)  from  the  water-supply  of  Berlin,  bears  a  close 
resemblance  to  the  cholera-bacillus  in  regard  to  form  and 
flagella.  Upon  gelatin-plates  the  border  of  the  colonies  is, 
however,  mostly  smooth.  The  colonies  themselves  exhibit  a 
much  more  finely  granular  appearance  than  those  of  the  comma- 
bacillus.     Gelatin  is  slowly  liquefied,  and  the  cholera-red  reac- 


408 


CLINICAL  BACTERIOLOGY. 


tion  appears.  Guinea-pigs  died  after  intraperitoneal  injection, 
with  precisely  the  same  symptom-complex  as  appeared  after 
introduction  of  true  comma-bacilli.  Similar  vibrios  have  been 
cultivated  from  Munich  well-water  by  Weibel;  from  Peene 
water,  by  Loffler ;  from  the  harbor  of  Groningen,  by  Fokker ; 
and  from  the  harbor  of  Altona,  by  Kiesling ;  and,  further,  from 
the  Seine.  In  forming  an  opinion  as  to  the  identity  of  these 
and  the  following  vibrios  with  the  true  exciting  agents  of  Asiatic 
cholera,  Pfeiffer's  reaction  (p.  187)  and  the  agglutination-test 
must  be  given  preeminent  importance.  Both  tests  yield  nega- 
tive results. 

J/.    Vibrio  Metschnikoff. — This  was  first  cultivated  in  an  epi- 
demic among  fowl,  and  subsequently  from  the  water  of  the  Spree. 


:^ 


Fig.  89.— Spirillum  berolinensis,  from  an  agar-agar  culture ;  X  looo  (Itzerott  and 

Niemann). 


It  is  somewhat  thicker  and  shorter  than  the  cholera-vibrio,  and 
sometimes  almost  coccus-shaped.  In  hanging  drop  it  exhibits 
active  motility.  The  cultures  resemble  those  of  the  cholera- 
bacillus,  except  that  liquefaction  is  more  marked.  The  nitroso- 
indol  reaction  appears  within  twenty-four  hours.  This  vibrio, 
in  contradistinction  from  that  of  cholera,  is  pathogenic  for  both 
pigeons  and  guinea-pigs. 

j<5*.  Vibrio  Gindha. — This  organism  was  cultivated  by  von 
Pasquale  from  well-water  in  Gindha  near  Massowah.  It  is  a 
rather  long,  slightly  curved  rod,  actively  motile  (with  a  single 
terminal  flagellum).  It  is  but  slightly  pathogenic,  and  it  does 
not  yield  the  nitroso-indol  reaction. 

jp.    Vibrio  Lissabon. — This  has  been  cultivated  in  the  course 


VIBRIOS  IN  WATER. 


409 


of  an  extensive   epidemic   of  cholerine   in  Lisbon,  in  which, 
however,  but  a  single  death  occurred.     On  gelatin-plates  cir- 


Fig.  90. — Spirillum  Metschnikoff,  from  an  agar-agar  culture ;  X  1000  (Itzerott  and 

Niemann). 


Fig.  91. — Spirillum  Metschnikoff;  stab-culture  in  gelatin  forty-eight  hours  old 
(Frankel  and  Pfeiffer). 


cular,  sharply  circumscribed,  whitish-yellow  colonies  form  that 
cause  little  liquefaction.  The  culture  does  not  yield  the  nitroso- 
indol  reaction. 


410  CLINICAL  BACTERIOLOGY. 

40.  Vibrio  Phosphorescens  Dunbar. — This  may  be  con- 
founded with  the  cholera-bacillus  on  account  of  its  morphologic 
and  cultural  appearances,  but  is  distinguishable  by  its  phos- 
phorescence. 

41.  Vibrio  Massowah. — This  possesses  from  two  to  four 
flagella,  whereas  the  comma-bacillus  possesses  but  a  single  ter- 
minal flagellum.  It  yields  the  nitroso-indol  reaction,  and  is 
quite  pathogenic  for  pigeons,  guinea-pigs,  and  rabbits. 


/'     V,      ,           .•*.-'•>    T- 

^  .^'  \.-r%''^  ^ 

\.\  :i.>.'rv.^  V     ;     -. 

""■^-.;*_  * 

Fig.  92.- 

—Spirillum  Dunbar,  from  agar-agar;  X  1000  (Itzerott  and  Niemann), 

n.  DISINFECTION. 

To  afford  protection  against  the  importation  of  epidemics, 
which  mostly  threaten  Europe  from  the  Orient,  resort  is 
had  to  supervision  of  maritime  intercourse  by  means  of 
guarantiite-stations,  the  International  Sanitary  Bureau,  and 
other  official  arrangements.  General  hygienic  efforts  also, 
through  drainage  of  the  soil,  through  the  provision  of 
a  pure  water-supply,  through  care  for  healthful  dwellings, 
etc.,  to  render  the  sanitary  conditions  of  the  country 
during  periods  free  from  pestilence  such  that  epidemics, 
when  imported,  do  not  find  the  soil  upon  which  they 
thrive,  must  be  made  by  the  public  authorities.  The  practis- 
ing physician  can  cooperate  in  these  to  only  a  slight  degree. 
The  conditions,  however,  are  different  when  once  the  epi- 
demic has  been  introduced  ;  then  the  relations  of  the  phy- 
sician with  regard  to  it  are  identical  with  those  that  exist 


DISINFECTION.  411 

with  regard  to  endemic  diseases,  such  as  typhoid  fever, 
whooping-cough,  tuberculosis,  etc.  These  also  may  attain 
epidemic  distribution.  The  duty  of  notification,  which  de- 
volves upon  the  physician,  renders  it  possible  for  the 
authorities  to  have  cognizance  of  every  extraordinary  in- 
crease in  the  prevalence  of  any  infectious  disease,  and  to 
recognize  the  epidemic  at  the  outset,  to  trace  its  causes 
and  eventually  to  remove  them.  An  important  part  of  the 
prophylactic  measures  against  disease  that  already  prevails 
lies  within  the  control  of  the  physician.  He  should  en- 
deavor to  prevent  the  increase  of  an  endemic  disease  to  the 
proportions  of  an  epidemic,  and,  when  the  disease  is  intro- 
duced from  without,  to  prevent  its  further  extension  by 
suppressing  the  source  of  infection,  by  rendering  the  indi- 
vidual case  harmless.  Disinfection,  the  destruction  of  dis- 
ease-germs, with  which  every  case  of  disease  threatens  its 
immediate  and  remote  environment,  is  an  integral  element 
of  all  disease-prophylaxis. 

In  the  preceding  chapters  it  has  been  pointed  out  in  de- 
tail how  the  germs  of  disease  pass  over  into  the  secretions 
and  excretions,  how  they  adhere  to  beds,  to  linen,  and  to 
sick-rooms,  etc.  It  has  also  been  pointed  out  how  they 
become  deposited  upon  articles  of  food  and  with  these  gain 
entrance  into  other  organisms.  All  of  these  carriers  of 
disease-germs  should  be  subjected  to  disinfection  in  every 
case  of  disease. 

The  agents  for  disinfection  act  mechanically  or  chemically. 
Those  of  the  first  group  are  aimed  at  the  removal  of  the 
disease-germs  mechanically  (by  brushing,  rubbing,  washing, 
rinsing,  scouring,  etc.),  or  at  their  destruction  directly  (by 
drying,  exposure  to  the  sun,  or  to  the  action  of  heat).  Of 
these  mechanical  means  the  first,  washing  with  brushes  and 
soap,  etc.,  are  in  common  employ.  Exposure  to  the  sun 
(airing  of  beds,  etc.)  is  probably  efficient,  but  can  influence 
only  the  bacteria  lying  superficially.  Heat  may  be  em- 
ployed in  the  form  of  hot  or  boiling  water,  of  hot  air,  and 
of  steam.  Boiling  water  is  an  active  disinfectant,  destroy- 
ing bacteria  generally  within  a  few  seconds,  and  particu- 
larly resistant  forms — apart  from  the  spores  of  harmless 
potato-bacilli  (mesentericus) — in  from  two  to  five  minutes. 
Hot  air  is  much  less  efficient.  According  to  Koch  and 
Wolff  hiigel,  the  most  resistant  of  the  spore-free  bacteria 
are  destroyed  by  exposure  for  one  and  a  half  hours  to  air 


412  CLINICAL   BACTERIOLOGY. 

at  a  temperature  of  80°  C.  (176°  F.),  and  spores  only  after 
exposure  for  three  hours  at  a  temperature  of  from  140°  C. 
(284°  F.  )  to  160°  C  (320°  F.).  Steam  may  be  employed 
still  or  streaming,  and  further  under  tension  and  super- 
heated. Live  steam  is  most  available  for  large  disinfecting 
apparatus.  These  represent  in  principle  nothing  more 
than  the  Koch  steam-chest.  Into  a  large  vessel  or  an  air- 
tight chamber,  steam -vapor  at  a  temperature  of  100°  C. 
(212°  F.)  is  so  introduced  that  it  displaces  all  of  the  air 
and  comes  in  contact  with  every  portion  of  the  articles  to 
be  disinfected.  As  in  the  steam-chest,  in  these  larger 
apparatus  also  complete  sterilization  is  effected  in  from 
fifteen  to  thirty  or  sixty  minutes,  in  accordance  with  the 
size  of  the  articles.  Steam  under  pressure  (120°  C.  —  248° 
F.)  is  employed  for  purposes  of  disinfection,  especially 
in  France.  With  its  aid  sterilization  is  effected  quickly 
and  with  absolute  certainty  when  provision  is  made  for  the 
escape  through  a  valve  of  all  the  air  contained  in  the  appa- 
ratus. 

Cold  inhibits  the  development  of,  but  scarcely  destroys, 
resistant  microorganisms.  Anthrax-spores  retain  their 
vitality  and  virulence  for  more  than  twenty-four  hours  at  a 
temperature  of  130°  C.  (266°  F.). 

Of  greater  importance  are  chemic  dismfectants.  These 
consist  of  all  sorts  of  chemic  agents  (acids,  alkalies,  salts, 
aromatic  substances,  etc.),  and  the  large  majority  cause 
enfeeblement  and  inhibit  the  development  of  the  bacteria, 
and  in  sufficient  concentration  effect  their  destruction. 

As  the  chemic  disinfectants  are,  in  the  nature  of  things, 
almost  invariably  employed  in  aqueous  solution,  recent  in- 
vestigations in  the  domain  of  physical  chemistry  with  regard 
to  the  nature  of  the  solutions  have  not  only  acquired  sig- 
nificance for  the  comprehension  of  observed  phenomena, 
but  they  have  also  yielded  valuable  aid  in  the  selection 
and  application  of  disinfectants.  In  the  course  of  these 
observations,  which  were  begun  by  Dreser,  then  continued 
especially  by  Paul  and  Kronig,  and  at  the  same  time  also 
by  Scheurlen  and  Spiro,  it  has  developed  that  the  changes 
which  the  dissolved  substances  undergo  in  the  solution 
through  the  action  of  the  solvent  are  of  decisive  import- 
ance with  regard  to  the  mode  of  action.  According  to  the 
views  of  Arrhenius,  now  almost  generally  accepted  as  cor- 
rect, a  dissociation  (division)  of  the  dissolved  salts,  acids, 


DISINFECTION.  413 

and  bases,  into  electropositive  and  electronegative  con- 
stituents— into  their  ions — takes  place  in  watery  solutions. 
While  in  general  the  salts  in  watery  solution  are  much 
more  actively  dissociated  than  the  acids  and  the  bases,  the 
degree  of  dissociation  of  the  various  salts  of  a  metal  is 
dependent  upon  the  nature  of  the  acid-ion,  and  that  of  the 
same  salt  in  turn  upon  the  dilution. 

As  the  chemic  and  physical  properties  of  solutions  are 
dependent  upon  the  degree  and  the  character  of  the  ioniza- 
tion, it  has  developed  that  also  the  disinfectant  activity  of 
equimolecular  solutions  is  influenced  by  the  dissociation, 
so  that,  for  instance,  solutions  of  mercurial  salts  are  the 
more  active  the  more  mercury  they  contain,  not  by  weight- 
percentage,  but  in  the  form  of  ions.  For  this  reason  solu- 
tions of  mercuric  chlorid  exceed  in  activity  solutions  of  all 
other  mercurial  salts. 

In  the  course  of  their  investigations  Paul  and  Kronig 
discovered  another  series  of  laws.  Thus,  the  acids  act  in 
general  accordance  with  their  electrolytic  dissociation  (ex- 
cept hydrofluoric  acid,  nitric  acid,  trichloracetic  acid),  and 
likewise  the  bases  in  accordance  with  the  concentration  of 
the  hydroxyl-ions  contained  in  the  solution.  It  has  also 
been  shown  that  the  oxidizing  agents — nitric  acid,  chromic 
acid,  chloric  acid,  persulphuric  acid,  and  permanganic  acid 
— are  active  in  accordance  with  the  position  that  they 
occupy  in  the  scale  of  oxidizing  agents  by  reason  of  their 
electric  activity.  The  halogens — chlorin,  bromin,  iodin — 
whose  disinfecting  activity  on  the  whole,  in  correspondence 
with  their  chemic  activity,  diminishes  with  increase  in  atomic 
weight,  play  a  specific  role. 

Whereas  ionization  has  thus  proved  itself  an  index  for 
both  the  chemic  and  the  disinfectant  activity  of  salts,  acids, 
and  bases — in  general,  the  inorganic  disinfectants — there 
exists,  according  to  Scheurlen  and  Spiro,  another  large 
group  of  disinfectants,  whose  activity  is  not  dependent  upon 
the  action  of  ions,  but  upon  **  molecular  action,"  and  in 
connection  with  which  the  entire  undissociated  molecule 
must  be  taken  into  consideration.  The  mode  of  action  of 
this  class  of  substances,  the  principal  representative  of 
which  is  carbolic  acid,  can  be  explained,  as  Spiro  has 
recently  shown,  by  analogy  with  the  laws  for  the  division  of 
a  body  between  two  solvent  agencies  :  that  is,  the  condi- 
tion present  is  not,  as  in  the  first  class  of  disinfectants,  one 


414  CLINICAL  BACTERIOLOGY. 

of  true  chemic  reaction,  but  one  of  solution  manifestations, 
similar  to  those  better-known  processes,  of  which,  for  in- 
stance, we  have  long  had  knowledge  in  a  whole  series  of 
stains. 

The  distinction  between  the  two  classes  of  disinfectants 
is  of  significance  also  for  practical  purposes.  It  thus  be- 
comes clear  why,  on  addition  of  other  substances — for 
instance,  acids — the  action  of  the  one  class  (chemic  disin- 
fectants) is  usually  lessened,  while  that  of  the  other — for 
instance  phenol — is  increased,  whereas  addition  of  alcohol 
has  the  reverse  effect.  An  explanation  is  afforded  also  for 
the  practically  significant  phenomenon  that  the  disinfection 
of  organic  or  albuminous  nutrient  media  (such  as  feces, 
sputum,  etc.)  is- effected  more  readily  with  the  second  class 
of  agents  than  with  the  first. 

In  order  to  determine  the  activity  of  a  disinfecting  agent, 
according  to  Koch's  procedure  highly  resistant  bacteria — 
anthrax-spores  that  have  been  dried  upon  short  pieces  of 
sterile  silk  thread  are  most  commonly  employed  as  test- 
objects — are  introduced  into  a  solution  of  the  agent  and 
from  time  to  time  it  is  noted  in  specimens  that  are  removed 
and  transferred  to  nutrient  medium  whether  the  bacteria 
have  already  been  destroyed  or  not.  Geppert  has  called 
attention  to  a  possible  source  of  error  attending  this  mode 
of  procedure.  With  the  silk  thread  not  alone  the  spores 
are  transferred  to  the  new  nutrient  medium,  but  always, 
even  when  the  thread  is  rinsed  with  water  after  removal, 
also  a  certain  amount  of  the  disinfecting  agent.  This 
diffuses  from  the  silk  into  the  nutrient  medium,  and  renders 
it  unsuitable  for  the  culture  of  the  bacteria.  Growth,  there- 
fore, may  readily  fail  to  take  place,  although  all  of  the  bac- 
teria may  not  be  destroyed. 

Koch  has  himself  studied  the  inhibition  of  growth  in 
culture-media  to  which  antiseptics  were  added  in  varying 
amount,  and  in  which  then  silk  threads  with  anthrax-spores 
were  introduced.      He  found 

Distinct  Retardation  Complete  Cessation 

OF  Growth  of  Growth 

On  Addition  of 
Mercuric  chlorid  in  the  strength  of  I  :  1,600,000  I  :  300,000 

Thymol  *'  I  :  80,000 

Oil  of  turpentine  **  1:75,000 

Potassium-soap  **  i  :  5000  i  :  looo 


DISINFECTION. 

415 

Distinct  Retardation 

Complete  Cessation 

OF  Growth 

On  Addition  of 

OF  Growth 

lodin                    in  the  strength  of  i  :  5000 

Salicylic  acid                      ** 

I  13300 

I  :  1500 

Hydrochloric  acid              ** 

I  :  2500 

I  :  1700 

Camphor                             *' 

I  :  2500 

More  than  i  :  1250 

Borax                                   " 

I  :  2000 

I  :  700 

Potassium  permanganate  '* 

I  :  1400 

Boric  acid                            " 

I  :  1250 

1:800 

Carbolic  acid                       " 

I  : 1250 

I  :850 

Quinin                                   *' 

I  :  830 

I  :  625 

Potassium  chlorate              " 

I  :  250 

Alcohol                                 " 

I  :  100 

1:12.5 

Sodium  chlorid                   *' 

I  :  64 

From  the  foregoing  it  will  appear  that  even  quite  slight 
additions  of  the  disinfectant  may  nullify  the  results  of  the 
experiment  when  the  method  of  investigation  first  named — 
of  introducing  the  silk  threads  into  the  nutrient  medium — is 
pursued.  When  Geppert,  by  means  of  dilute  solutions  of 
ammonium  sulphate,  removed  completely  the  disinfectant 
from  the  anthrax-spores  that  had  lain  in  mercuric-chlorid 
solution,  I  :  1000,  before  their  transference  to  the  nutrient 
medium,  he  still  observed  growth  after  exposure  to  the 
action  of  the  mercuric  chlorid  for  fifteen  minutes,  and  also 
after  exposure  for  five  hours,  and  in  one  instance  even  after 
exposure  for  twenty-four  hours.  The  spores  were  still 
infective.  Animals  that  Geppert  inoculated  therewith  fre- 
quently died  of  anthrax.  Solutions  of  mercuric  chlorid  of 
I  :  100  did  not  with  certainty  destroy  the  spores  in  from 
six  to  twelve  minutes.  From  this  it  can  be  seen  that  the 
disinfecting  activity  of  the  agents  tested  by  the  method  of 
Koch  has  probably  been  often  placed  too  high.  The 
figures  obtained  by  Koch  and  numerous  subsequent  investi- 
gators still  retain  their  value  for  practical  purposes,  as  Koch 
himself  endeavored  to  reduce  to  a  minimum  the  sources 
of  error  indicated  by  making  the  silk  threads  as  short, 
and  using  as  large  an  amount  of  culture-medium,  as 
possible,  and  rinsing  in  distilled  water,  alcohol,  etc.,  the 
threads  removed  from  the  antiseptic  solution  before  trans- 
ference to  the  nutrient  solution. 

The  following  table  from  Fliigge's  "  Text-book  "  (1897) 


416 


CLINICAL  BACTERIOLOGY. 


will  give  an  idea  of  the  utility  of  the  better  known  anti- 
septics : 


Destroy 

Strepto- 

Anthrax-bacilli, 

Bactericidal 

cocci  AND 
Staphylo- 
cocci. 

Typhoid-bacilli, 
Cholera-bacilli. 

Agents. 

Anthrax-spores. 

Within 
Five  Min- 
utes. 

Within  Five 
Minutes. 

In  from  Two 
to  Twenty- 
four  Hours. 

Hydrogen  dioxid     . 

Concen- 
trated 

I  :200 

1:500 

1: 100  after  an  hour. 

Chlorin 

O.I    per 

O.I     per 

... 

Fresh  chlorin- water 

cent. 

cent. 

0.2  per  cent,  in 
an  hour. 

lodin  trichlorid    .    . 

I  :  200 

I  :  1000 

Potassium  iodid    .    . 

... 

I  :  10 

Sulphuric  or  hydro- 

chloric acid  .    .    . 

I  :  10 

I  :  100 

1:1500;  ty- 
phoid, 1:700 

I  :  50  in  ten  days. 

Sulphurous  acid   .    . 

I  :  300,  gas 
10   vol.    per 

cent,  (only 
superficially) 

Arsenic  acid .... 

... 

... 

... 

I  :  1000  after  ten 
days. 

Boric  acid 

... 

... 

1:30 

Concentrated  after 
six  days  incom- 
pletely. 

Potassium  hydroxid . 

1:5 

... 

I  :300 

Ammonia 

... 

I  :300 

Soda 

... 

... 

I  :40 

Ammonium   carbon- 

ate       

... 

... 

I  :  100 

Calcium  hydroxid    . 

... 

I  :  1000 

Silver  nitrate     .    .    . 

I  :  1000 

... 

I  :4000 

Mercuric  chlorid  .    . 

I :  10,000 
-I : 1000 

I  :2000 

... 

I  :2000 

Copper  sulphate   .    . 

... 

... 

... 

I  :  20  (five  days). 

Potassium     perman- 

ganate    

I  :200 

... 

... 

I  :  20  (one  day). 

Potassium  bichromate 

... 

... 

... 

I  : 1700 

Chlorinated  lime  .    . 

I  :500 

I  :  20  (one  hour). 

Ferric  chlorid   .    .    . 

... 

... 

I  :  20  (six  days). 

Alcohol 

80  per  ct. 

... 

... 
... 

Acetic    acid,    oxalic 

acid,  etc 

1 :  200  or  300 

Chloroform   .... 

I  :  14 

Carbolic  acid    .    .    . 

iViSo 

Choi  era, 
I  :  200; 

glanders, 

anthrax, 
I  :  100; 

typhoid, 
1:50 

I  :  300 

1 :  20  in  from  four  to 
forty-five  days. 

DISINFECTION. 


417 


Destroy 

Bactericidal 
Agents. 

Strepto- 
cocci AND 
Staphylo- 
cocci. 

Anthrax-bacilli, 
Typhoid-bacilli, 
Cholera-bacilli. 

Anthrax-spores. 

Within 
Five  Min- 
utes. 

Within  Five 
Minutes. 

In  from  Two 
to  Twenty- 
four  Hours. 

Salicylic  acid     .    .    . 
Creosote    .    .    .    .    . 
Creosote  sulphate     . 
Creolin 

Aseptol 

Quinin 

Oil  of  turpentine  .    . 

I  :  lOOO 
1:300 

1  :  500 

I  :  100 

From  3  to  5 
per  cent. 

1:3000;  ty- 
phoid, 1 :  250 

I  :  20  in  six  hours. 

10  per  cent,  in 
thirty  minutes. 

1: 100  after  ten  days. 

Concentrated  in  five 
days. 

Upon  the  basis  of  a  comparison  of  the  results  of  Koch's 
observations  with  those  of  numerous  subsequent  investiga- 
tors Schimmelbusch  arranges  the  disinfectants  in  accord- 
ance with  the  time  in  which  they  destroy  anthrax-spores,  in 
the  following  order : 


II. 


Mercuric  chlorid,  \ 

lodid,  chlorin,  and  bromin,  /  j    ^  ^i,  •*!,• 

lodin  trichlorid  (Behring),  7enT-four^hours 

Kresol  rendered  soluble  by  addition  j  ^* 

of  sulphuric  acid  (C.  Frankel),    / 

Five  per  cent,  carbolic  acid,  creolin,  1  j    .        .1  •      u     4.4       j 

Crude  wood-vinegar,  |  ^^^^^^^  ^^^  ^P^''^^  ^"  ^^^"^  *^°  ^^y'' 

Chlorinated  lime,  five  per  cent,      ^ 

Oil  of  turpentine,  f 

Ammonium  sulphate. 

Formic  acid, 

Ferric  chlorid,  five  per  cent., 

Chlorpicrin,  five  per  cent. , 

Quinin,  one  per  cent.,  with  hydro- 
chloric acid, 

Arsenic  acid,  one  in  the  thousand,  i 

Hydrochloric  acid,  two  per  cent.,  ) 

Ether  destroys  the  spores  in  about  thirty  days. 
III.  Anthrax-spores  are  still  not  destroyed  after  the  lapse  of  a  month  in  absolute 
alcohol,  distilled  water,  chloroform,  glycerin,  benzoic  acid,  ammonia, 
concentrated  solution  of   sodium  chlorid,  five  per  cent,   potassium 
chlorate,  alum,  borax. 


destroy  the  spores  in  about  five  days, 
destroy  the  spores  in  about  six  days, 
y  destroy  the  spores  in  about  ten  days. 


The  figures  obtained  experimentally,  however,  constitute 
no  trustworthy  index  of  the  practical  utility  of  an  agent. 
For  practical  purposes  the  greater  or  lesser  solubility  of  a 
27 


418  CLINICAL  BACTERIOLOGY. 

disinfectant,  its  capacity  for  penetrating  the  objects  to  be 
disinfected — for  this  reason  oily  solutions  of  even  active  an- 
tiseptics are  almost  ineffective,  because  the  oil  does  not 
penetrate  into  the  organisms — its  chemic  constitution  (upon 
which  is  dependent  the  extent  to  which  the  object  to  be 
disinfected  suffers  in  the  process  of  disinfection),  and  much 
besides  must  be  taken  into  consideration.  The  cost  of  a  dis- 
infecting agent  is  also  not  without  bearing  upon  its  practical 
applicability  upon  a  large  scale. 

It  would  carry  us  too  far  to  discuss  individually  the  in- 
numerable antiseptics  that  have  been  tested  in  recent  years, 
and  that  have  been  recommended  for  one  purpose  or 
another.  We  shall  limit  ourselves  to  the  following  obser- 
vations : 


DISINFECTION  OF  THE  HANDS. 

Disinfection  of  the  hands  is  practised  at  surgical  clinics 
in  the  following  manner,  in  accordance  with  the  regulations 
of  Fiirbringer  :  The  hands  are  washed  as  clean  as  possible 
in  tepid  water  with  soap  and  vigorous  brushing  for  five 
minutes,  then  rinsed  in  fresh  water ;  the  nails  are  cleaned, 
especially  the  spaces  beneath  the  nails,  by  means  of  a  metal- 
lic nail-cleaner ;  the  hands  are  then  rubbed  in  alcohol  for 
three  minutes  ;  next  they  are  rinsed  and  rubbed  for  a  minute 
in  from  ^  to  i  :  looo  solution  of  mercuric  chlorid.  In  the 
event  of  marked  contamination  of  the  skin,  it  is  first  rubbed 
with  ether  before  the  disinfectants  are  used,  or  the  entire 
procedure  is  performed  twice. 

Before  every  operative  procedure  this  process  of  rigid 
disinfection  must  be  carried  out  completely  at  the  site  of 
operation,  as  well  as  on  the  hands  of  the  operator.  A  simpler 
mode  of  disinfection  of  the  hands — ^washing  with  soap  and 
brushing,  rinsing  with  mercuric  chlorid  or  alcohol — should 
be  practised  by  physicians  and  attendants  always  after  con- 
tact with  a  patient,  and  especially  before  each  meal.  In 
houses  in  which  an  infectious  disease  exists,  especially  in 
times  of  epidemic,  disinfection  of  the  hands  before  eating  is 
a  general  duty.  Instead  of  mercuric  chlorid,  the  following 
may  also  be  employed  :  Carbolic  acid,  from  3  to  5  per 
cent.  ;  creolin,  3  per  cent.  ;  lysol,  i  ^  per  cent.  The  sub- 
stances last  named,  however,  have,  as  has  been  pointed 
out,  less  disinfecting  power  than  mercuric  chlorid. 


DISINFECTION.  419 

DISINF5CTION  OF  MUCOUS  MEMBRANES. 

The  disinfection  of  mucous  membranes  is  far  more  diffi- 
cult than  that  of  the  external  integument.  Simple  irriga- 
tion with  a  disinfectant  will  not  yield  the  desired  result — 
quite  apart  from  the  danger  of  intoxication.  Vigorous 
brushing,  however,  and  alcohol  and  ether  are  naturally 
not  applicable  here  in  the  same  degree  as  upon  the  exter- 
nal integument.  The  greatest  stress  is,  therefore,  to  be 
placed  upon  mechanical  removal  of  the  germs,  and  this  may 
be  effected  by  brushing  and  rubbing  with  the  fingers  or 
with  swabs  of  cotton  or  of  gauze.  Irrigation  of  the  loos- 
ened mucus  and  debris  may  be  practised  with  simple  warm 
water,  or  with  nonirritant  solutions  (weak  solutions  of 
boric  acid,  potassium  permanganate,  aluminum  sulphate, 
physiologic  salt-solution,  infusion  of  chamomile,  etc.). 


DISINFECTION  OF  INSTRUMENTS  AND  DRESSINGS. 

Metallic  instruments  are  boiled  for  five  minutes  in  water, 
preferably  after  addition  of  one  per  cent,  of  soda,  whereby 
rusting  is  avoided.  Cotton,  gauze  bandages,  and  the  like 
are  sterilized  in  the  steam-chest  or  the  dry  chamber. 


DISINFECTION  OF  FECES  AND  CESSPOOLS, 

Lime  is  best  adapted  for  disinfection  of  the  stools.  In 
the  bed-pan  in  which  the  infectious  stool  (especially  in 
cases  of  typhoid  fever  and  of  cholera)  is  received,  so  much 
milk  of  lime  is  previously  introduced  as  will  just  cover  the 
bottom  of  the  vessel.  After  defecation  an  amount  of  milk 
of  lime  is  added  equal  to  the  bulk  of  the  feces,  and  the 
mixture  is  vigorously  agitated  and  permitted  to  stand  for 
an  hour.  The  mixture  must  be  highly  alkaline  (Pfuhl). 
The  milk  of  lime  should  always  be  freshly  prepared  :  To 
unslaked  lime  in  stone  jars  or  wooden  pails  so  much  water 
is  added  as  will  be  taken  up.  The  slaked  lime  is  diluted 
with  four  times  the  amount  of  water. 

If  chlorinated  lime  be  employed,  it  must  be  added  in  an 
amount  equal  to  one  per  cent,  of  the  mixture  of  urine  and 
feces.     It  may  be  added  in  the  form  of  powder  or  of  a  paste 


420  CLINICAL  BACTERIOLOGY. 

consisting  of  about  twenty  grams  of  chlorinated  lime  and 
lOO  of  water.  After  thorough  admixture  the  stool  need 
stand  for  only  fifteen  minutes. 

For  the  disinfection  of  cesspools  Pfuhl  likewise  recom- 
mends milk  of  lime.  When  the  tun-system  is  used,  3  grams 
=  6  cu.  cm.  of  pulverulent  slaked  lime,  or,  better,  30  cu. 
cm.  of  milk  of  lime,  and  in  the  case  of  pits  2  grams  =  4 
cu.  cm.  of  pulverulent  lime,  or,  better,  20  cu.  cm.  of  milk 
of  lime  should  be  allowed  daily  for  each  person.  The 
seat  as  well  as  the  funnel-shaped  basin  and  the  discharge -pipe 
must  be  thoroughly  irrigated  with  the  disinfecting  solution. 
If,  in  addition  to  feces,  which  may  be  estimated  at  400  cu. 
cm.  for  each  person  daily,  also  the  total  amount  of  urine  is 
thrown  into  the  receptacle,  the  mass  of  material  to  be  dis- 
infected may  be  estimated  at  between  1 500  and  2000  cu. 
cm.,  and  four  or  five  times  as  much  of  the  disinfectant 
should  be  employed  for  each  person.  Under  these  conditions 
also  disinfection  is  attained  only  when  the  privy-contents 
exhibit  a  distinctly  alkaline  reaction. 

Of  other  disinfectants  for  water-closets,  carbolic  acid, 
sulphocarbolic  acid,  lysol,  and  saprol,  may  be  mentioned. 
These  must  be  introduced  in  solution,  and  in  an  amount 
equal  to  two  per  cent,  of  the  material  to  be  disinfected ; 
about  eight  grams  daily  may,  therefore,  be  estimated  for 
each  person.  All  of  these  substances  are  more  expensive 
than  milk  of  lime,  without  possessing  especial  advantages. 
For  hospitals  it  may  be  recommended  that  the  entire  fecal 
accumulation  be  boiled  in  a  suitable  vessel  with  addition  of 
deodorizing  substances  (potassium  permanganate). 


DISINFECTION  OF  BATH-WATER,  WASH-WATER,  ETC 

Bath-water,  when  contaminated  by  patients,  is  disinfected 
by  means  of  milk  of  lime  (six  liters  to  a  bath  of  300  liters) 
or  of  carbolic  acid.  Mercuric  chlorid  is  to  be  avoided 
when  metallic  tubs  are  used. 

Wash-water  is  freed  from  typhoid-bacilli  and  cholera- 
bacilli,  according  to  Pfuhl,  within  an  hour  if  i.  5  to  one  thous- 
and of  calcium  hydroxid  be  added,  and  if  constant  agitation 
is  maintained. 

Koch  permitted  milk  of  lime  to  be  introduced  into  the 
drainage-fluid  at  Nietleben  until  the  fluid  appearing  in  the 


DISINFECTION.  421 

main  discharge -conduit  at  the  lower  end  of  the  irrigation- 
field  presented  a  marked  alkaline  reaction. 

For  the  disinfection  of  water-conduits  dilute  milk  of  lime, 
carbolic  acid,  or  a  mineral  acid,  is  employed.  At  Nietleben 
Koch  disinfected  the  service  by  means  of  carbolic  acid,  per- 
mitting three  per  cent,  carbolic  acid  to  be  driven  from  the 
pumping  well  into  all  divisions  of  the  service,  and  to  remain 
in  the  conduits  for  twenty-four  hours.  Then  the  pipes 
were  irrigated  with  wholesome  water.  This  procedure  is 
attended  with  the  objection  that  the  water  retains  for  a  con- 
siderable time  the  taste  of  carbolic  acid.  On  the  other 
hand,  however,  it  does  not  carry  with  it  the  danger  of  ob- 
struction of  the  conduits  that  attends  the  employment  of 
milk  of  lime. 


DISINFECTION  OF  URINEL 

The  urine  is  usually  disinfected  in  common  with  the  feces. 
It  is  generally  not  so  infective  as  the  stools.  If  it  is  to  be 
disinfected  alone,  this  may  be  accomplished  by  addition  of 
milk  of  lime,  carbolic  acid,  or  mercuric  chlorid. 


DISINFECTION  OF  SPUTUM. 

The  sputum  must  be  received  and  preserved  in  a  moist 
condition,  preferably  in  sputum-cups  containing  a  layer  of 
water.  So  long  as  the  sputum  is  contained  within  such 
cups  it  is  relatively  harmless.  A  greater  danger  resides  in 
the  conversion  into  dust  and  into  spray  of  sputum  evacu- 
ated into  handkerchiefs,  upon  floors,  etc.  On  emptying 
the  vessel  the  sputum  must  be  disinfected.  Crude  carbolic 
acid  (from  5  to  10  per  cent.)  or  mercuric  chlorid  (i  or  2  : 
1000)  would  be  suited  for  this  purpose  if  the  disinfectant 
penetrated  the  sputum.  Generally,  however,  the  albumin 
on  the  outer  surface  of  the  mass  of  sputum  is  coagulated, 
and  the  bacilli  contained  within  do  not  come  in  contact  with 
the  antiseptic  at  all.  For  this  reason  the  sputum  must  at 
least  be  thoroughly  rubbed  up  in  the  disinfecting  solution, 
and  remain  therein  for  a  long  time.  Lysol  (10  per  cent.) 
and  crude  solutol  (from  5  to  10  per  cent.)  do  not  coagulate 
the  sputum,  and  are  therefore  better  adapted  for  disinfec- 
tion.    It  is  further  useful  to  disinfect  the  sputa  by  heat.    If 


422  CLINICAL  BACTERIOLOGY. 

they  are  not  too  abundant  and  are  at  the  same  time  viscid, 
they  can  be  simply  burned  in  a  stove.  Otherwise  they  are 
introduced,  together  with  the  sputum-cup,  into  a  spe- 
cially constructed  disinfector,  resembling  the  steam-chest 
(Kirchner),  in  which  they  are  exposed  for  half  an  hour  to 
the  action  of  steam  at  a  temperature  of  ioo°  C.  (212°  F.). 
The  apparatus  has  the  disadvantage  that  a  number  of  the 
cups  will  be  broken,  and  in  families  it  can  not  often  be  pro- 
vided. Under  such  circumstances  the  sputa  can  be  poured 
into  a  pot  and  boiled  for  half  an  hour  in  water.  Usually, 
however,  one  will  have  to  be  satisfied  with  simply  emptying 
them  into  the  water-closets,  where  the  pathogenic  germs 
are  simultaneously  destroyed,  in  part  with  the  disinfection 
of  the  feces  and  in  part  by  the  process  of  putrefaction.  The 
sputum-cups  should  be  sterilized  by  means  of  hot  water  and 
carbolic  acid,  and  preferably  likewise  boiled. 


DISINFECTION  OF  BODY-CLOTHING  AND  BED-LINEN. 

Uncontaminated  linen  is  boiled  for  half  an  hour  in  petro- 
leum soap-water  (two  bucketfuls  of  water — about  thirty 
liters — to  250  grams  of  soft  soap  and  two  spoonfuls  of  petro- 
leum ;  then,  after  removal  of  the  soap-water,  rinsed  in  cold 
water ;  next  washed  with  soap  in  clean  hot  water  ;  then 
again  rinsed  in  cold  water,  permitted  to  remain  over  night 
in  clean  water,  and  finally  dried  in  the  open  air.  Instead 
of  this  procedure  the  linen  may  also  be  disinfected  in  the 
steam  disinfecting  apparatus. 

Contaminated  linen  requires  removal  of  feces,  mucus,  or 
pus  before  application  of  heat,  as  otherwise  burned  spots 
will  appear.  Such  linen  must  immediately  after  removal  be 
placed  in  a  sheet  moistened  with  mercuric  chlorid,  i  :  2000, 
then  in  strong,  moist  sacks,  and  sent  away  for  disinfection. 
The  sacks,  unopened,  are  introduced  into  the  disinfecting 
fluid,  three  per  cent,  soft-soap  solution,  in  which  the  linen  is 
treated  for  three  hours  at  a  temperature  of  50°  C.  (122°  F.), 
and  then  remains  for  an  additional  forty-eight  hours  during 
the  process  of  cooling  ;  or  mercuric-chlorid  salt  solution 
(of  0.5  to  one  thousand  mercuric  chlorid  and  6  to  one 
thousand  sodium  chlorid).  After  disinfection  in  this  way 
the  linen  is  further  treated  in  the  same  manner  as  uncon- 
taminated linen. 


DISINFECTION.  423 


DISINFECTION  OF  BEDS  AND  CLOTHING. 

Articles  of  clothing  and  beds  are  best  disinfected  in  suit- 
able apparatus  in  live  steam.  If  such  an  apparatus  is  not 
available,  exposure  to  air  and  sun  must  be  resorted  to  or 
rubbing  with  three  per  cent,  carbolic  acid.  To  purify  arti- 
cles by  exposure  to  air,  they  must  be  hung  for  days  in  a 
dry  room  and  exposed  as  uniformly  as  possible  upon  all 
sides  to  the  rays  of  the  sun.  Even  then  the  disinfection  is 
not  trustworthy.  The  bedstead,  articles  of  leather,  and  the 
like,  are  rubbed  off  with  five  per  cent,  carbolic  acid.  For 
the  disinfection  of  clothing  formalin  (a  forty  per  cent,  solu- 
tion of  formaldehyd)  has  been  warmly  recommended. 
The  clothing  should  be  loosely  packed  in  a  chest,  and  be- 
tween its  layers  are  placed  strips  of  goods  saturated  with  for- 
malin. From  thirty  to  fifty  grams  of  formalin  are  required  for 
a  suit  of  clothing.  Disinfection  is  said  to  be  completed 
within  two  hours,  and  the  disagreeable  odor  may  be 
removed  by  means  of  ammonia. 


DISINFECTION  OF  ARTICLES  OF  FOOD. 

The  keeping  clean  of  articles  of  food  is  especially  a  pro- 
phylactic measure.  These  should  not  be  permitted  to  stand 
about  in  the  sick-room,  but  they  should  be  kept  covered, 
etc.  Portions  of  food  left  unused  by  the  patient  are  burned, 
as  well  as  articles  of  food  that  are  known  to  have  been  in- 
fected in  other  ways.  Milk  and  other  liquids  are  drunk 
only  boiled,  as  should  also  be  drinking-water  in  times  of 
cholera-epidemic. 


DISINFECTION  OF  THE  SICK-ROOM. 

A  sick-room  should  not  contain  pictures,  curtains,  etc., 
in  fact  any  superfluous  articles  that  only  serve  as  dust-col- 
lectors. After  the  termination  of  the  illness  the  room  of 
the  patient,  and  possibly  the  entire  dwelling,  should  remain 
undisturbed  for  about  ten  hours,  to  permit  the  dust  to  settle, 
and  then  it  should  be  thoroughly  disinfected.  In  some  large 
cities  this  is  undertaken  by  special  establishments.  Such 
disinfecting  stations  should  be  established  everywhere,  for 


424  CLINICAL  BACTERIOLOGY. 

only  with  their  aid  and  through  their  trained  attendants  can 
thorough  disinfection  of  dweUings  be  carried  out.  All 
portable  articles  that  will  withstand  the  action  of  moist 
steam  are  wrapped  in  cloths  moistened  with  three  per  cent, 
carbolic  acid  or  mercuric  chlorid,  i  :  looo,  and  sent  to  the 
disinfecting  station,  where  they  are  placed  in  a  steam-appa- 
ratus. The  walls  and  ceilings  are  thoroughly  rubbed  down 
with  bread-crumbs,  and  these  are  eventually  burned  in  the 
stove.  Walls  painted  with  oil-colors  are  washed  with  five 
per  cent,  carbolic  acid  or  painted  with  milk  of  lime.  White- 
washed walls  are  given  a  new  coat.  The  furniture  is 
rubbed  off  with  three  per  cent,  carbolic  acid  and  then  rubbed 
dry.  Polished  articles  can  be  rubbed  off  with  bread.  Up- 
holstered furniture,  when  possible,  is  sterilized  in  the  steam- 
apparatus  ;  otherwise  rubbed  down  with  carbolic  acid  and 
brushed.  Articles  of  leather,  metal,  glass,  and  the  like  can 
be  vigorously  rubbed  with  carbolic  acid.  Mantels,  the 
upper  surfaces  of  stoves,  etc.,  are  first  freed  of  dust  by  means 
of  moist  cloths,  then  soaped  and  rubbed  with  three  per  cent, 
carbolic  acid.  Finally,  the  floor  is  disinfected  by  scouring 
with  warm  water  and  soap  and  then  with  carbolic  acid.  Of 
late  formaldehyd,  which  has  already  been  mentioned  as  a 
disinfectant  for  clothing,  has  also  been  employed  for  the  dis- 
infection of  dwellings.  The  following  mode  of  procedure 
is  pursued  :  A  forty  per  cent,  aqueous  solution  of  formal- 
dehyd (also  known  as  formalin  or  formol)  is  mixed  with 
chlorinated  lime  and  water — to  each  liter  of  formalin  200 
grams  of  chlorinated  lime  in  400  cu.  cm.  of  water ;  the 
mixture  is  designated  formochloral — and  the  mixture  is 
evaporated  in  a  Trillat  autoclave,  with  four  atmospheres  of 
pressure.  One  liter  of  formochloral  is  sufficient  for  200 
cubic  meters  of  air-space.  The  disinfection  is,  however, 
essentially  only  superficial,  the  bacteria  in  thick  articles, 
such  as  beds,  mattresses,  clothing,  etc.,  not  being  destroyed 
thereby.  After  disinfection  has  been  completed  the  vapor 
of  formaldehyd  is  removed  by  means  of  a  spray  of  ammonia 
and  exposure  to  air.  Greater  advantages  than  the  Trillat 
apparatus,  whose  manipulation  is  always  difficult,  are  pos- 
sessed by  the  formalin-lamp  placed  upon  the  market  by 
Schering,*  with  the  aid  of  which  the  disinfecting  vapor  is 
generated  in   a  most  simple   manner  by  the  burning   of 

*  Numerous  forms  of  serviceable  apparatus  can  now  be  obtained. 


DISINFECTION.  425 

formalin-pastils.  Two  pastils,  each  of  one  gram,  are  em- 
ployed for  every  cubic  meter  of  space.  According  to  our 
experience,  trustworthy  superficial  disinfection  can  be 
effected  by  this  means  for  all  bacteria  without  spores. 


DISINFECTION  OF  SHIPS,  VEHICLES,  RAILWAY-CARS,  ETC. 

Entire  ships,  cars,  etc.,  are  disinfected  in  a  manner  similar 
to  that  employed  in  disinfection  of  the  sick-room.  The 
burning  of  sulphur,  which  was  formerly  much  practised  (20 
grams  of  sulphur  to  each  cubic  meter  of  space,  moistened 
with  alcohol  before  ignition),  has  been  shown  by  Koch's 
investigations  to  be  relatively  useless,  and  injures  many 
articles  rather  seriously. 


INDEX 


Abscess,  127 

complicating  typhoid  fever,  127 

local,  123 

of  liver,  155,  370 

pyemic,  155 

tropical,  155 

Abscesses,  cold,  127 

Absolute  immunity,  56 

Achorion,  cultural  properties  of,  342 

Schonleinii,  341 
Acquired  immunity,  50 
Actinomyces,  aerobic,  360 

botanic  position  of,  361 

pure  culture  of,  357 
Actinomyces-druses,  356 
Actinomyces-granules,  356 
Actinomycosis,  354 

bacteriologic  diagnosis  of,  362 

course  of,  357 

experimental  development  of,  360 

in  human  beings,  355 

of  animals,  354 

treatment  of,  362 
Active  immunization,  55,  68 
Aerial  germs,  sources  of,  394 
Aerobic  bacteria,  26 
Agar  streak-plates,  94 
Agar-agar,  plates  of,  94 

preparation  of,  81 
Agar-plates,  95 
Agglutination,  62 
Agglutination-phenomenon  in  typhoid 

fever,  176 
Air,  bacteria  in,  393,  401 

bacteriologic  examination  of,  389, 
392 

exclusion  of,  anaerobic  pure  culture 
with,  loi 

germs  in,  sources  of,  394 

Hesse's  procedure  for  examination 
of,  392 


Air,  hot,  disinfection  by,  41 1 
method  of  examination  of,  392 
Petri's  procedure  for   examination 
of,  392 

Air-infection,  394 

Albuminoids,  culture-media  free  from, 

87 
Alexins,  60 
Amebse,  examination   of  stools   for, 

371 
in  normal  intestinal  contents,  371 
Amebic  enteritis,  364 
Amount  of  infectious  material,  39  ^ 
Amphitrocha,  19 
Anaerobic  bacteria,  26 
cultivation  of,  98 
facultative,  26 
plate-culture  of,  98  ' 
pure    culture    by   the   method   of 
Buchner,  loi 
in  a  high  layer,  loi 
in  an  atmosphere  of  hydrogen, 

loi 
in  test-tubes,  loi 
raw  eggs  for,  102 
with   complete    exclusion    of 
air,  loi 
Angina,  134 

bacteriologic  diagnosis  of,  135 
due  to  mixed  infection,  135 
herpetica,  129 
prognosis  of,  135 
scarlatinal,  135 
Anginas,  phlegmonous,  134 
pseudo-  membranous,   1 34 
Aniline  water  as  a  mordant,  104 
Animal   experimentation,  determina- 
tion of  pathogenicity  (or   speci- 
ficity) of  bacteria  by,  no 
life,  infections  with  lowest  forms  of, 

364 
Anthrax,  287 

bacteriologic  diagnosis  of,  298 


427 


428 


INDEX. 


Anthrax,  experimental    development 
of,  295 

heredity  of,  297 

immunity  to,  299 

mixed  infection  in,  297 

occurrence  of,  in  animals,  291 
in  human  beings,  292 

prophylaxis  of,  300 

vaccination  against,  299 
Anthrax-bacilli,  cultivation  of,  289 

distribution    of,    in  infected  body, 
296 

infection    with,    through    digestive 
tract,  293 
through  kmgs,  295 
through  skin,  293 

morphology  of,  287 

natural  portals  of  entry  for,  293 

resistance  of,  290 

sporulation  of,  290 

toxins  of,  297 
Anthrax-carbuncle,  292 
Anthrax-spores,  destruction  of,  by  dis- 
infectants, 417 

resistance  of,  290 
Anti-bodies,  59 
Antiseptics,  utility  of,  416 
Antitoxic  power  of  blood-serum,  70 
Antitoxin -immunity,  71 
Antitoxins,  67 
Arthrosporous  bacteria,  23 
Artificial  immunity,  50 
Ascending  pyelonephritis,  159 
Aseptic  peritonitis,  151 
Asiatic  cholera,  181 
Aspergilli,  336 
Aspergillus  flavescens,  339 

fumigatus,  339 
Attenuation-method  of  immunization, 

54 


Babes-Ernst  bodies,  19 
Bacillary  filaments,  20 
Bacilli,  17 

Bacillus  aerogenes,  122 
amethystinus,  404 
aquatilis,  404 
arborescens,  402 
aurantiacus,  401 
botulinus,  245 

cultural  peculiarities  of,  246 
morphology  of,  246 
occurrence  of,  250 
pathogenic  properties  of,  247 
tenacity  of,  247 
brunneus,  402 
coeruleus,  404 


Bacillus  constrictus,  401 

fluorescens  liquefaciens,  402 

nonliquefaciens,  401 
immobilis,  401 
fuscus,  401 
helvolus,  403 
ianthinus,  403 
liquefaciens,  404 
liquidis,  404 
mesentericus,  405 
pavoninus,  404 
prodigiosus,  403 
pyocyaneus,  120,  125,  126 
rubefaciens,  402 
ruber  aquatilis,  404 

balticus,  404 
rubidus,  403 
spinosus,  405 
subflavus,  402 
subtilis,  404 
violaceus,  403 
viscosus,  403 
Bacteria,  aerobic,  26 
anaerobic,  26 

cultivation  of,  98 
arthrosporous,  23 
biology  of,  17 
chemic  activities  of,  28 

combinations  due  to,  26 
classification  of,  17 
determination  of  pathogenicity  (or 

specificity)  of,  by  animal  experi- 
mentation, no 
diagnostic  significance  of  metabolic 

products  of,  28 
endosporous,  23 
facultative  anaerobic,  26 
ground-substance  of,  19 
identification  of,  28 
in  soil,  air,  and  water,  401 
influence  of  oxygen  on,  26 
light  in  relation  to,  26 
metabolic  products  due  to,  29 
microscopic  examination  of,  102 
morphology  of,  17 
multiplication  of,  20 
nonpathogenic,  33 
pathogenic,  23 
permanent  forms  of,  22,  23 
pigment  formed  by,  26 
pleomorphic,  24 
rod- shaped,  17 
spheric,  17 
spiral,  17 
staining  of,  102 
sustenance  of,  25 
thermophilic,  22 
ubiquity  of,  25 
Bacteriacese,  17 


INDEX. 


429 


Bacterial   diseases,    susceptibility  to, 

45 

peritonitis,  151 

poison,  specific,  30 

proteids,  31 
Bactericidal  substances,  59 
Bacterium  coli  commune,   120,   125, 
126 
differentiated     from    typhoid- 
bacilli,  174 

encapsulated,  19 

lactis  aerogenes,  126 
Basidia,  337 

Bath-water,  disinfection  of,  420 
Bed-linen,  disinfection  of,  422 
Beds,  disinfection  of,  423 
Beggiatoa,  27 
Biliary  abscesses,  155 
Birth,  infection  during,  49 
Blood,  typhoid-bacilli  in,  171 
Blood-agar,  preparation  of,  82 
Blood- serum  agar,  84 

antitoxic  power  of,  70 

glycerin-agar,  84 

human,  preparation  of,  84 

liquid,  preparation  of,  83 

of  immunized  animals,   immuniza- 
tion with,  54 
Bodies,  permanent,   22 
Body-clothing,  disinfection  of,  422 
Bone-marrow,  typhoid-bacilli  in,  170 
Botulism,  245 

bacteriologic  diagnosis  of,  250 

immunity  to,  251 

mixed  infection  in,  250  ' 

physiology  of,  249 

specific  therapy  of,  251 
Bouillon,  preparation  of,  78 
Bread-pap,  86 
Bronchitis,  138 

fetid,  138 
Brush-molds,  337 
Buchner,    method  of,    for   anaerobic 

pure  culture,  loi 
Budding  fungi,  338 

infections  with,  336 
Bulbous  molds,  336 


C. 

Capsules,  staining  of,  108 

Carbolic  acid  as  a  mordant,  104 

Carbuncle,  126 

Causative  agents  of  inflammation  in 
disease,  126 
in  healthy  persons,  126 
morphology  of,  115 
outside  the  body,  126 


Causative    agents    of    inflammation, 
pathogenic  properties  of,  with 
relation  to  animals,  123 
of  suppuration   in   healthy  per- 
sons, 125 
outside  the  body,  125 
Causes  of  immunity,  57 
Cavities  of  the  body,  inoculation  of, 

III 
Cell-capsule,  19 
Cell-membrane,  19 
Cesspools,  disinfection  of,  419 
Chain,  closed,  extension  of  cholera  in, 
190 

continuous,  disease-extension  by,  47 
Chain -cocci,  20 
Chalazion,  1 61 
Chemic  activities  of  bacteria,  28 

combinations  due  to  bacteria,  26 

disinfection,  412 
Chemotaxis,  negative,  58 

positive,  58 
Chickenpox,  328 
Cholangitis,  154 

bacteriologic  diagnosis  of,  155 

experimental  development  of,  155 
Cholecystitis,  154 

bacteriologic  diagnosis  of,  155 

experimental  development  of,  155 
Cholera,  Asiatic,  181 
cause  of,  181 

bacteriologic  diagnosis  of,  193 

development  of,  188 

disinfection  of,  196 

epidemics  of,  190 

experimental  development  of,  191 

explosive  extension  of,  190 

extension  of,  in  closed  chain,  190 

immunity  to,  197 

local  predisposition  to,  191 

nostras,  198 

prophylaxis  of,  196 

temporal  predisposition  to,  19I 

toxins  of,  189 
Cholera-bacilli,  181 

cultural  investigation  of,  193 

examination  of  water  for,  195 

gelatin  plate -culture  of,  193 

microscopic  examination  of,  193 

peptone-culture  of,  194 
Cholera-red  reaction,  184 
Cholera-vibrios,  occurrence  of,  1 86 

tenacity  of,  185 
Chorea,    endocarditis    secondary    to, 

148 
Choroiditis,  160 
Cicatrix-tetanus,  239 
Circumscribed  peritonitis,  152 
Clostridium,  23 


430 


INDEX. 


Clothing,  disinfection  of,  423 

Coccacese,  17 

Cocci,  17 

Cold  abscesses,  128 

action  of,  on  bacteria,  412 
Columella,  337 

Comma-bacilli,  appearance  of,  in  cul- 
tures, 182 
Condensation,  water  of,  82 
Congenital  pneumonia,  148 

syphilis,  319,  320 

tuberculosis,  268 
Conidia,  336 
Conjunctivitis,  160 
Contagion,  ^^  ' 

Contagious  disease,  ^^ 

miasmatic  disease,  34 
Continuous    chain,   disease-extension 

by,  47 
Cover-glass  specimen,  preparation  of, 
104 
staining  of,  104 
Crescents,  Laveran's,  373 
Cryptogenetic  septicemia,  162 
Cultivation  of  anaerobic  bacteria,  98 
Culture,  methods  of,  74,  87 

pure,  74,  87 
Culture-media  free  from  albuminoids, 

87 
Cure,  49 

immunity  and,  relations  between, 

71 

Cutaneous  inoculation,  no 
Cystitis,  155 

bacteriologic  diagnosis  of,  156 

experimental  development  of,  156 

gonorrheal,  155 

puerperal,  155 

tuberculous,  155 


D. 

Death-point,  thermal,  22 
Dermatomycoses,  341 
Descending  pyelonephritis,  159 
Diagnostic  significance  of  metabolic 

products  of  bacteria,  28 
Diarrhea,  summer,  199 
Diffuse  peritonitis,  152 
Digestive    tract,    infection    with    an- 
thrax-bacilli through,  293 
typhoid  fever  through,  169 
peritonitis  arising  from,  15 1 
Dilution-method  of  immunization,  54 
Diphtheria,  207 

bacteriologic  diagnosis  of,  217 

immunity  to,  218 

mixed  infection  in,  213 


Diphtheria,  physiology  of,  in  animals, 
211 

prophylaxis  of,  230 

relations  of  diphtheria-bacillus  to, 
213 

specific  therapy  of,  218 

susceptibility  of  human  beings  to, 
215 
Diphtheria-antitoxin,      complications 
from  use  of,  229 

methods  of  testing,  225 
Diphtheria-bacillus,  207 

cultural  properties  of,  209 

pathogenic  properties  of,  2IO 

portal  of  infection  for,  216 

relations  of,  to  diphtheria,  213 

saprophytic  occurrence  of,  214 

staining  properties  of,  207 

tenacity  of,  210 
Diphtheria-toxin,  active,  220 
Diphtheric  endocarditis,  148 
Diplobacilli,  21 
Diplobacillus  Friedlander,  126 

pneumoniae  Friedlander,  119,  125 
Diplococci,  21 
Diplococcus  lanceolatus  Frankel,  124 

pneumoniae  Frankel,  118,  126 
Direct  immunization,  55,  68 

infection,  48 
Discontinuous  sterilization,  77 
Disease,  causative  agents  of  inflam- 
mation in,  126 

contagious,  33 
miasmatic,  34 

explosive  distribution  of,  47 

infectious,  33 

miasmatic,  33 
Disease-extension  by  continuous 

chain,  47 
Disinfectant,  activity  of,  414 
Disinfection,  410 

by  boiling  water,  41 1 

by  heat,  411 

by  hot  air,  41 1 

by  hot  water,  411 

by  steam,  411 

chemic,  412 

in  cholera,  196 

mechanical,  411 

of  bath-water,  420 

of  bed-linen,  422 

of  beds,  423 

of  body-clothing,  422 

of  cess- pools,  419 

of  clothing,  423 

of  dressings,  419 

of  feces,  419 

of  food,  423 

of  hands,  418 


INDEX. 


431 


Disinfection  of  instruments,  419 
of  mucous  membranes,  419 
of  railway-cars,  425 
of  ships,  425 
of  sick-room,  423 
of  sputum,  421 
of  urine,  421 
of  vehicles,  425 
of  wash- water,  420 
of  water-conduits,  421 
Double  staining,  107 
Dressings,  disinfection  of,  419 
Drinking-water,    infection    with    ty- 
phoid fever  through,  170 
Drumstick-bacteria,  22 
Dysentery,  364 
amebse  of,  364 
death  of,  366 

in    the   stools  of  dysenteric  pa- 
tients, 367 
movement  of,  365 
nutrition  of,  365 
pathogenic  activity  of,  368 
permanent  forms  of,  366 
portals  of  infection  for,  369 
propagation  of,  366 
resistance  of,  366 
staining  of,  367 
transmission  of,  369 
intestinal  alterations  of,  367 
occurrence  of  bacteria  in,  368 


E. 

Ecthyma,  129 
Ectoplasm,  364 
Edema,  malignant,  305 
bacillus  of,  305 

cultural  properties  of,  306 
occurrence  of,  307 
bacteriologic  diagnosis  of,  308 
experimental     development    of, 

307 
immunity  to,  308 
in  human  being,  308 
portal  of  infection  for,  308 
Eggs,  raw,  for  anaerobic  culture,  102 
Eisner's  modification  of  potato-gela- 
tin, 81 
Endemic  occurrence  of  infectious  dis- 
eases, 47 
Endocarditis,  148 

bacteriologic  diagnosis  of,   150 

complicating  acute  nephritis,  149 

diphtheric,  148 

experimental  development  of,  149 

gonorrheal,  149 

malignant,  149 


Endocarditis,  secondary,  148 

to  acute  articular  rheumatism,  147 

chorea,  148 

croupous  pneumonia,  148 

erysipelas,  148 

erythema  nodosum,  148 

influenza,  148 

osteomyelitis,  148 

puerperal  fever,  148 

pyemia,  148 

scarlet  fever,  149 

septicemia,  148 

smallpox,  149 

suppurative  processes,  148 
tuberculous,  149 
typhoid,  149 
ulcerative,  149 
Endometritis,  159 
Endosporous  bacteria,  23 
Enteritis,  amebic,  364 
Entoplasm,  364 
Epidemic  distribution  of  pneumonia, 

147 

of  typhoid  fever,  1 70 
occurrence  of  infectious  diseases,  47 

Epidemics  of  cholera,  190 
of  disease,  47 

Erysipelas,  129 

bacteriologic  diagnosis  of,  13I 
bullosum,  130 

endocarditis  secondary  to,  148 
etiologic  significance  of  streptococ- 
cus for,  131 
immunity  to,  131 
microorganisms  in,  131 
pericarditis  secondary  to,  150 
phlegmonous,  130 
specific  treatment  of,  132 

Erythema  nodosum,  endocarditis  sec- 
ondary to,  148 

Erythrasma,  348 

Esmarch's  modification  of  Koch's 
procedure,  94 

Examination,  methods  of,  74 

Exanthemata,  acute,  328 

Experimentation,  animal,  determina- 
tion of  pathogenicity  (or  specificity) 
of  bacteria  by,  no 

Explosive  distribution  of  disease,  47 
extension  of  cholera,  190 

Eye,  anterior  chamber  of,  inoculation 
of.  Ill 
inflammatory  diseases  of,  160 


Farcy,  304 
Favus,  341 


432 


INDEX. 


Favus,  diagnosis  of,  344 
Feces,  disinfection  of,  419 

tubercle-bacilli  in,  264 

typhoid-bacilli  in,  171 
Fetid  bronchitis,  138 
Fibrinous  rhinitis,  133 
Filamentous    fungi,    infections   with. 

Filaments,  bacillary,  20 
Filtration  of  water,  399 
Fission-fungi,  17,  336 
Fixed  phagocytes,  58 
Flagella,  19 

staining  of,  109 
Food,  disinfection  of,  423 

infection      with       typhoid      fever 
through,  169 
Fowl -tuberculosis,  275 
bacillus  of,  275 
diagnosis  of,  276 

experimental  development  of,  276 
occurrence  of  bacilli  of,  276 
Fractional  sterilization,  77 

streak,  95 
Fragmentation,  337 
Fruit-bearer,  336 
Fungi,  budding,  338 
biology  of,  336 
cultivation  of,  339 
diseases  due  to,  341 
infections  with,  336 
microscopic  examination  of,  338 
morphology  of,  336 
pathogenic  activity  of,  339 
filamentous,  biology  of,  336 
diseases  due  to,  341 
infections  with,  336 
microscopic  examination  of,  338 
morphology  of,  336 
pathogenic  activity  of,  339 
Furuncle,  126 


G. 

Gall-bladder,  peritonitis  arising  from, 

151 

Gas-abscesses,  127 

bacteriologic  investigation  of,  128 

Gastro-enteritis,  hemorrhagic,  311 

Gastro-intestinal   lesions,    abscess  of 
liver  secondary  to,  155 
tract,  inoculation  through.  III 

Gelatin,  preparation  of,  80 

General  predisposition,  45 

Genital  organs,  female,  inflammations 
of,  159 

Genitourinary  tract,  female,  peritoni- 
tis arising  from,  151 


Giant-ameba,  364 
Glanders,  300 

bacteriologic  diagnosis  of,  304 

clinical  picture  of,  304 

course  of,  303 

experimental  development  of,  302 

heredity  of,  304 

portals  of  entry  for,  303 

prophylaxis  of,  305 

susceptibility  of  animals  to,  302 
Glanders-bacilli,    cultural    properties 
of,  301 

distribution  of,  in  products  of  dis- 
ease, 302 

morphology  of,  300 

occurrence  of,   in  products  of  dis- 
ease, 302 

resistance  of,  302 
Globular  molds,  337 
Gonococci,  cultivation  of,  for  diagnos- 
tic purposes,  313 

culture  of,  312 

double  staining  of,  313 

morphology  o^  311 

occurrence  of,  311 

specific  pathogenic  significance  of, 

Gonorrhea,  311 

bacteriologic  diagnosis  of,  313 
prophylaxis  of,  314 
Gonorrheal  cystitis,  155 

endocarditis,  149 
Gram's  method  of  staining,  106 

Giinther's   modification    of, 

107 
Weigert's   modification    of, 
107 
Growth,  variability  in  form  of,  25 
Gruber's  reaction,  63 
Giinther's    modification    of    Gram's 
method  of  staining,  107 


H. 

Hands,  disinfection  of,  418 
Hay,  infusion  of,  d)"] 
Hay-bacillus,  404 

Healthy  persons,  causative  agents  of 
inflammation  in,  125 
of  suppuration  in,  1 25 
Heat,  disinfection  by,  41 1 

dry,  74  . 
Hemoglobin-agar,  82 
Hereditary  syphilis,  319 

transmission  of  pneumonia,  147 
Heredity  of  anthrax,  297 

of  glanders,  304 

of  immunity,  56 


INDEX. 


433 


Heredity  of  infectious  diseases,  48 

of  leprosy,  280 

of  syphilis,  319 

of  tuberculosis,  267 
Herpes,  129 

labialis,  129 

of  larynx,  129 

of  pharynx,  129 

tonsurans,  344 

zoster,  129 
Hesse's  procedure  for  examination  of 

air,  392 
High   layer,  anaerobic   pure    culture 

in,  loi 
House-epidemics  of  pneumonia,  147 
House-filters,  400 
Hydrogen,  atmosphere  of,  anaerobic 

pure  culture  in,  loi 
Hydrophobia,  321 

experimental  development  of,   321 

immunization  to,  ^^,  323 

incubation  of,  322 

results  of   Pasteur's  treatment   of, 

3'f5  .  .. 
susceptibility  to,  321 
vaccination  against,  323 
Hyph?e,  336 


I. 

Ice,  4CX) 

artificial,  400 

natural,  400 
Icterus,  febrile,  310 
Identification  of  bacteria,  28 
Idiopathic  tetanus,  239 
Immediate  immunization,  55,  68 
Immunity,  49 

absolute,  56 

acquired,  50 

and  cure,  relations  between,  71 

artificial,  50 

causes  of,  57 

heredity  of,  57 

natural,  49 

to  measles,  328 

quantitative  limitations  of,  56 

reaction  of,  66 

relative,  56 

specificity  of,  56 

to  anthrax,  299 

to  botulism,  251 

to  cholera,  197 

to  diphtheria,  218 

to  erysipelas,  131 

to  influenza,  286 

to  malignant  edema,  308 

to  plague,  206 
28 


Immunity  to  smallpox,  326 

to  syphilis,  318 

to  tetanus,  241 

to  typhoid  fever,  179 
Immunization,  49 

active,  55,  68 

attenuation-method  of,  54 

dilution-method  of,  54 

direct,  55,  68 

immediate,  55,  68 

indirect,  55,  68 

mediate,  55,  68 

methods  of,  50 

passive,  55,  68 

to  hydrophobia,  323 

with    blood- serum    of    immunized 
animals,  54 
Immunization-therapy,  72 
Impetigo,  128 

Indirect  immunization,  55,  68 
Infected   organism,   susceptibility  of, 

44 
Infection,  23^  3^ 

direct,  48 

during  birth,  49 

infectious,  34 

intrauterine,  49 

mixed,  41 

period  of,  reaction  of,  66 

portals  of,  42 

secondary,  46 

toxic,  34 
Infections  with  budding  fungi,  336 

with  filamentous  fungi,  336 

with    lowest  forms  of  animal  life, 

364 
Infectious  agent,  virulence  of,  37 
disease,  S3 

diseases,  endemic  occurrence  of,  47 
epidemic  occurrence  of,  47 
heredity  of,  48 

pneumonia  complicating,  145 
infection,  34 
material,  amount  of,  39 
purity  of,  39,  41 
Inflammation,  115 

causative    agents    of,    in     disease, 
126 
in  healthy  persons,   125 
morphology  of,  115 
outside  the  body,   125 
pathogenic     properties     of, 
with  relation  to  animals, 
123 
local,    123 
Inflammations   of   female  genital  or- 
gans,  159 
Inflammatory  diseases  of  the  eye,  160 
Influences,  predisposing,  46 


434 


INDEX. 


Influenza,   281 

bacteriologic  diagnosis  of,  287 

endocarditis  secondary  to,  148 

experimental  development  of,  286 

immunity  to,  286 
Influenza-bacillus,  cultivation  of,  281 

distribution  of,  285 

localization  of,  285 

morphology  of,  280 

occurrence  of,  284 

resistance  of,  284 

staining  of,  281 
Influenza-pneumonia,  145 
Inhalation,  inoculation  by,  ill 
Injection,  intravenous,  no 
Inoculation  by  inhalation,  in 

cutaneous,  no 

of  the  anterior  chamber  of  the  eye, 
III 

of  the  cavities  of  the  body,  in 

protective,  against  plague,  206 

subcutaneous,  no 

subdural,  in 

through  gastro-intestinal  tract,  in 
Instruments,  disinfection  of,  429 
Intestinal  mycosis,  295 
Intoxication,  putrid,  due  to  proteus, 
310 

typhoid,  172 
Intrauterine  infection,  49 
Intravenous  injection,  no 
Involution-changes,  25 
Irids,  160 


K. 

Keratitis,  160 
Keratomycoses,  349 
Kidneys,  typhoid-bacilli  in,  171 
Koch  procedure,  Esmarch's  modifica- 
tion of,  94 
Koch's  method  of  plate-making,  88 


Laryngitis,  acute,  133 
Larynx,  herpes  of,  129 
Laveran's  crescents,  373 
Leprosy,  278 

bacillus  of,  278 

bacteriologic  diagnosis  of,  280 

distribudon  of,  279 

experimental  development  of,  279 

heredity  of,  280 

prophylaxis  of,  280 
Leptothrix  buccalis,  348 
Leydenia  gemmipara  Schaudinn,  387 
Light  in  relation  to  bacteria,  26 


Limitations,   quantitative,    of   immu- 
nity, 56 
Liver,  370 

abscess  of,  155 

bacteriologic  diagnosis  of,  155 
examination  of  pus  from,  370 

abscesses  of,   secondary  to  gastro- 
intestinal lesions,  155 

peritonitis  arising  from,  151 

pyemic  abscesses  of,  155 

tropical  abscess  of,  155,  364 

typhoid-bacilli  in,  170 
Local  abscess,  123 

inflammation,  123 

predisposition,  45 
Localization,  secondary,  46. 
Lophotrocha,  19 

Lungs,   infection  with  anthrax-bacilli 
through,  295 
with  typhoid  fever  through,  169 

typhoid-bacilli  in,  171 
Lustgarten,  bacillus  of,  314 
Lymphangitis,  132 
Lysogenic  activity  of  immune  serum, 

62 
Lyssa,  321 


M. 

Macrophages,  58 
Malaria,  372 

exciting  agent  of,  372 
Malarial  fever,  continued,  381,  382 
double  quartan,  378 

tertian,  380 
explanation  of  symptoms  of,  by 

presence  of  parasites,  384 
false  quotidian,  380 
irregular,  378,  380,  381,  382 
malignant  tertian  parasite  of,  381 
mode  of  infection  with,  385 
parasites  of,  372 

biology  of,  372 

classification  of,  377 

development  of,  375 

morphology  of,  372 

multiplicity  of,  376 

polymorphism  of,  376 

relation  of,  to  red  blood-cor- 
puscles, 375 

sporulation  of,  375 
period  of  incubation  of,  386 
prognosis  of,  384 
quartan  parasite  of,  377 
quotidian  parasite  of,  380 
severe  tertian,  382   * 
spontaneous  recovery  from,  386, 

387 
tertian  parasite  of,  378 


INDEX. 


435 


Malarial  fever,  triple  quartan,  378 
typical  quartan,  378 
quotidian,  381 
tertian,  380 
infection,  mixed,  382 
parasites,  diagnosis  of,  382 

in  soil,  391 
pigment,   373 
Malignant  edema,  305 

bacillus  of,  in  soil,  39I 
endocarditis,  149 
pustule,  292 
Mallein,  305 
Malt-gelatin,  81 
Measles,  328 

contagium  of,  328 
endocarditis  following,  I49 
immunity  to,  329 
infectivity  of,  329 
natural  immunity  to,  328 
Meat-poisoning,  245 

with      gastrointestinal     symptoms, 

245 
Mediate  immunization,  55,  68 
Melanin,  373 

Meninges,  typhoid-bacilli  in,  171 
Meningitis,   136 

bacteriologic  diagnosis  of,  137 

clinical  diagnosis  of,  137 

epidemic  cerebrospinal,  136 

metastatic,  136 

primary,  136 

secondary,  136 
Mesenteric  glands,  typhoid-bacilli  in, 

170 
Metabolic  products  due    to  bacteria. 

Metastatic  pleurisy,  139 
Miasm,  34 

Miasmatic  disease,  ^^ 
Micrococci,  17 
Micrococcus  agilis,  406 

aquatilis,  406 

aurantiacus,  405 

candicans,  406 

concentricus,  406 

cremoides,  406 

radiatus,  406 

rosettaceus,  406 

versicolor,  405 
Microphages,  58 
Microscopic  examination  of  bacteria, 

102 
Microsporon  furfur,  347 

minutissimum,  348 
Milk,  86 

tubercle-bacilli  in,  265 
Mixed  infection,  41 
Mobile  phagocytes,  58 


Molds,  336 

bulbous,  336 

cultivation  of,  339 

globular,  337 

segmented,  338 
Monotrocha,  19 
Morphology  of  the  causative  agents 

of  inflammation,  1 15 
Mucor  corymbifer,  339 

rhizopodifoi-mis,  339 
Mucorini,  337 
Mucous  membranes,  disinfection  of, 

419 
Multiple  purulent  foci,  123 
Mycelium,  336 
Mycoderma  vini,  338 
Mycoses,  336 

visceral,  350 
Mycosis,  intestinal,  295 

pharyngis  leptothricia,  348 
Myocarditis,  151 

acute  diffuse,  15 1 

suppurative,  15 1 
Myocardium,  typhoid-bacilli  in,  1 71   » 
Myringomycoses,  349 


N. 

Natural  immunity,  49 

predisposition,  44 
Negative  chemotaxis,  58 
Nephritis,  157 

acute,  157 

endocarditis  complicating,  149 

bacteriologic  diagnosis  of,  158 

complicating,  157 
Nitrification,  391 
Nitrobacteria,  27 
Nitrosobacteria,  27 
Nitrosococcus,  27 
Nitroso-indol  reaction,  122 
Nitrosomonas,  27 
Noma,  133 

Nonpathogenic  bacteria,  ;^^ 
Nose,  inflammations  of,  133 
Notification,  41 1 
•Nutrient  media,  preparation  of,  78 


Oidia,  338 

Oidium  albicans,  351,  353 

Oophoritis,  159 

Ophthalmia,  sympathetic,  161 

Organism,  infected,  susceptibility  of, 

44 
Osteomyelitis,  164 


436 


INDEX. 


Osteomyelitis,  endocarditis  secondary 

to,  148 
experimental  development  of,  164 
Otitis  media,  135 

bacteriologic  diagnosis  of,  136 
Otomycoses,  349 

Oxygen,  influence  of,  on  bacteria,  26 
Ozena,  133 

P. 

Panaris,  127 

Pandemics  of  disease,  47 
Parametritis,  160 
Parasites,  ^^ 

facultative,  ^3 
Passive  immunization,  55,  68 
Pasteur's  treatment  of  hydrophobia, 

323 
results  of,  325 
Pathogenic  bacteria,  33 

properties   of  causative    agents   of 
inflammation    with    relation    to 
animals,  123 
Pathogenicity  of  bacteria,  deteimina- 
tion  of,  by  animal  experimentation, 
1 10 
Penicillii,  337 
Peptone-water,  86 
Perforative  peritonitis,  152 
Pericarditis,  150 

bacteriologic  diagnosis  of,  15 1 
primary,  150 
puerperal,  150 
pyemic,  150 
secondary,  150 

to    acute   articular    rheumatism, 

150 
to  erysipelas,  150 
to  pneumonia,  150 
traumatic,  150 
tuberculous,  150 
Perinephritis,  158,  160 
Peritonitis,  151 

arising   from    the    digestive    tract, 

151 

the  female  genito-urinary  tract, 

151 

the  gall-bladder  and  liver,  15 1 
aseptic,  151 
bacterial,   151 

bacteriologic  diagnosis  of,  152 
circumscribed,  152 
diffuse,  152 

due  to  operative  intervention,  152 
of  hematogenous  origin,  152 
perforative,  152 
septic,  151 
Peritrocha,  19 


Perityphlitis,  154 
Permanent  bodies,  22 

forms  of  bacteria,  22,  23 
Pertussis,  330 
Petri's  procedure  for  examination  of 

air,  392 
Peyer's   patches,    typhoid-bacilli    in, 

170 
Pfeiffer's  reaction,  62 
Phagocytes,  fixed,  58 

mobile,  58 
Phagocytic  theory,  58 
Phagocytosis,  36 
Pharyngitis,  acute,  133 
Pharyngomycoses,  348 
Phlebitis,  132 
Phlegmonous  anginas,  134 

erysipelas,  1 30 
Phlegmons,  putrid,  fetid,  due  to  pro- 
teus,  310 

urinary,  127 
Pigment  formed  by  bacteria,  26 

malarial,  373 
Pigment-bacteria,  26 
Pityriasis  versicolor,  347 
Plague,  200 

bacteriologic  diagnosis  of,  204 

etiologic  relations  of  plague-bacilli 
to,  204 

experimental  development  of,  203 

immunity  to,  206 

prophylaxis  of,  205 

protective  inoculation  against,  206 
Plague-bacilli,  appearance  of,  in  cul- 
ture, 201 

etiologic    relations    of,    to  plague, 
204 

mode  of  distribution  of,  202 

morphology  of,  200 

portals  of  infection  for,  202 

vital  capability  of,  20I 
Plasma,  granular,  364 
Plate-cocci,  21 
Plate-culture    of   anaerobic  bacteria, 

98 
Plate-making,  Koch's  method  of,  88 
Plates  of  agar-agar,  94 
Pleomorphic  bacteria,  24 
Pleural    effusions,  tubercle-bacilli    in 

fluid  from,  264 
Pleurisy,  metastatic,  139 

primary,  139 

secondary,  139 
Pleuritis,  138 

bacteriologic  investigation  of,  140 

diagnostic    and  prognostic   signifi- 
cance  of    bacteriologic   findings 
in,  140 
Pneumaturia,  156 


INDEX. 


4.;7 


Pneumococcus-angina,  134 
Pneumococcus-pneumonia,  143 
Pneumonia,  142 

bacteriologic  diagnosis  of,  145 
complicating     infectious     diseases, 

145 
tuberculosis,  145 

congenital,  148 

croupous,    endocarditis    secondary 
to,  148 

direct  transmission  of,  147 

epidemic  distribution  of,  147 

hereditary  transmission  of,  147    . 

house-epidemics  of,  147 

mixed  forms  of,  144 

pericarditis  secondary  to,  150 

prognostic  significance  of  bacterio- 
logic findings  in,  146 

transmission  of,  147 
Pneumonomycoses,  150 
Pneumothorax,  142 
Poison,  specific  bacterial,  30 
Portals  of  infection,  42 
Positive  chemotaxis,  58 
Potassium    hydroxid   as   a   mordant, 

104 
Potato-bacillus,  405 
Potatoes,  preparation  of,  84 
Potato-gelatin,  81 

Eisner's  modification  of,  81 
Predisposing  influences,  46 
Predisposition,  44 

general,  45 

local,  45 

temporary,  44 

to  smallpox,  326 
Preparation  of  cover-glass  specimens, 

104 
Primary  pericarditis,  150 

pleurisy,  139 
Prophylactic  inoculations  against  te- 
tanus, 244 
Prophylaxis  of  anthrax,  300 

of  cholera,  196 

of  diphtheria,  230 

of  glanders,  305 

of  gonorrhea,  314 

of  leprosy,  280 

of  plague,  205 

of  tuberculosis.  266 
Proteids,  bacterial,  31 
Proteus,  cultural  properties  of,  309 

febrile  icterus  due  to,  310 

hemorrhagic  gastro-enteritis  due  to, 

3" 

morphology  of,  308 
occurrence  of,  310 
putrid,    fetid   phlegmons    due    to, 
310 


Proteus,  putrid  intoxication  due  to,  310 
Proteus-infection,  bacteriologic  diag- 
nosis of,  311 

experimental  development  of,  310 
Proteus-infections,  308 
Protozoa,  364 
Prunes,  decoction  of,  87 
Pseudo-diphtheria-bacilli,   214 
Pseudo-filaments,  20 
Pseudo-influenza-bacilli,  287 
Pseudo-membranous  anginas,  134 
Pseudo-tuberculosis,  276 

bacterial,  277 

due  to  animal  parasites,  276 
to  foreign  bodies,  276 
to    highly    organized    vegetable 
parasites,  278 

etiology  of,  276 
Psoriasis,  348 
Ptomains,  28 
Puerperal  cystitis,  155 

fever,  163 

endocarditis  secondary  to,  148 

pericarditis,  150 
Pure  culture,  87 
Purity  of  infectious  material,  39 
Purulent  foci,  multiple,  123 
Pus  from    cold    abscesses,    tubercle- 
bacilli  in,  264 

tubercle-bacilli  in,  263 
Pustule,  malignant,  292 
Pyelonephritis,  159 

ascending,  159 

bacteriologic  diagnosis  of,  159 

descending,  159 

experimental  development  of,  159 
Pyemia,  123,  161 

bacteriologic  diagnosis  of>  162 

endocarditis  secondary  to,  148 

experimental  development  of,   163 
Pyemic  abscesses  of  the  liver,  155 

pericarditis,  150 
Pyocyaneous  general    infection,    165 


Q. 

Quartan  parasites  of  malaria,  377 
Quinin,  action  of,  386 


R. 

Rabies,  321 

resistance  of  virus  of,  323 
Rag-pickers'  disease,  295 
Railway-cars,  disinfection  of,  425 
Ray-fungus,  356 

portal  of  infection  for,  350 


438 


INDEX. 


Reaction,  Gruber's,  63 
of  immunity,  66 
of  infection,  66 
Pfeiffei's,  62 
Relapsing  fever,  332 

occurrence  of  spirilla  of,  333 
recovery  from,  334 
spirilla  of,  332 
transmission  of,  334 
Relative  immunity,  56 
Rheumatic  tetanus,  239 
Rheumatism,    acute   articular,    endo- 
carditis secondary  to,  148 
pericarditis  secondary  to,   150 
articular,  331 
Rhinitis,  acute,  133 

fibrinous,  133 
Rhinoscleroma,  133 
Rice-pap,  87 
Rod-shaped  bacteria,  17 
Roll-plate,  94 
Root-bacillus,  404 


S. 

Saccharomyces  cerevisise,  338 

hominis,  363 

subcutaneus  tumefaciens,  363 
Salpingitis,  159 
Sand-filtration  of  water,  399 
Saprophytes,  S3 

facultative,  33 
Sarcina  lutea,  406 

yelloM^,  406 
Sarcinse,   21 
Sausage-poisoning,   245 
Scarlatinal  angina,   135 
Scarlet,  fever,  329 

endocarditis  following,   149 
incubation  of,   329 
virus  of,  329 
Scrofulosis,  258 
Secondary  endocarditis,   148 

infection,  46 

localization,  46 

pericarditis,   150 

pleurisy,   139 
Sections  of  tissue,  staining  of,  106 

tubercle-bacilli  in,   264 
Segmentation,  337 
Segmented  molds,  338 
Self-purification  of  water,   398 
Sepsis,   123 
Septic  peritonitis,  151 
Septicemia,  34,   123,   161 

bacteriologic  diagnosis  of,   1 62 

cryptogenetic,  162 

endocarditis  secondary  to,  148 


Septicemia,     experimental     develop- 
ment of,  163 
Serum,    immune,    lysogenic    activity 

of,  62 
Serum-immunity,  71 
Serum-plates,  preparation  of,  83 
Serum-therapy,  72 

of  tetanus,  242 
Ships,  disinfection  of,  425 
Sick-room,  disinfection  of,  423 
Skin,    infection    with    anthrax-bacilli 
through,  293 

parasitic  diseases  of,  34I 
Smallpox,  325 

endocarditis  following,    149 

immunity  to,  326 

portal  of  infection  for,  325 

predisposition  to,   326 

virus  of,   325 
Soil,  bacilli  in,  390 

bacillus   of   malignant    edema    in, 

391 
of  tetanus  in,   390 
bacteria  in,  390,  401 
bacteriologic  examination  of,   389 
malarial  parasites  in,  391 
method  of  investigation  of,  389 
permanent  spores  in,   391 
Specific  therapy,   72 
of  botulism,  251 
of  diphtheria,   218 
Specificity  of  bacteria,  determination 
of,  by  animal  experimentation, 
no 
of  immunity,  56 
Spheric  bacteria,  17 
Spiral  bacteria,  17 
Spirilla,  17 
Spirillaceae,  17 
Spleen,  puncture  of,  in  typhoid  fever, 

175 

typhoid-bacilli  in,  170 
vSporangium,  337 
Spore-formation,  23 

arthrogenous,  23 

endogenous,  23 
Spore-germination,  23 
Spore-membrane,  22 
Spores,  22,  23,  336 

staining  of,  108 
Sputum,  disinfection  of,  42I 

tubercle-bacilli  in,  263 
Stab-culture,  96 
Stained  preparations,  examination  of, 

103 
Staining,  double,  107 

Gram's  method  of,  106 

Giinther's  modification  of  Gram's 
method  of,  107 


INDEX. 


439 


Staining  of  bacteria,  I02 

of  capsules,  io8 

of  cover-glass  specimens,  104 

of  flagella,  109 

of  sections  of  tissue,  106 

of  spores,  108 

Weigert's  modification  of  Gram's 
method  of,  107 
Staphylococci,  21,  124,  125 
Staphylococcus  cereus  albus,  117 
flavus,  117 

pyogenes  albus,  1 16 
aureus,  115 
citreus,  1 16 
Staphylococcus-angina,  134 
Staphylococcus-pneumonia,  144 
Steam,  disinfection  by,  41 1 

live,  sterilization  by,  75 
Sterigmata,  337 
Sterilization,  74 

by  dry  heat,  74 

by  live  steam,  75 

discontinuous,  77 

fractional,   77 
Streak,  fractional,  95 
Streak-culture,  96 
Streak-plates,  agar,  94 
Streptococci,  20,  124,  125 

microscopic     arrangement    of,     in 
skin,  130 
Streptococcus  brevis,  118 

conglomeratus,  118 

etiologic  significance  of,  for  erysip- 
elas, 131 

lanceolatus  Pasteur,  118 

longus,    118 

pyogenes  (erysipelatis),  117 
Streptococcus-angina,  134 
Streptococcus-diseases,  specific  treat- 
ment of,  132 
Streptococcus-pneumonia,  144 
Streptothricege,'24 
Streptothrices,  337 

pathogenic,  362 
Streptothnx  Eppinger,  362 

farcinica,  363 
Subcutaneous  inoculation,  no 
Subdural  inoculation,  in 
Sulphur-bacteria,  27 
Summer  diarrhea,  190 
Suppuration,  115 

causative  agents  of,  in  healthy  per- 
sons, 125 
outside  the  body,  125 
Suppurative  myocarditis,  151 

processes,    endocarditis    secondary 
to,  148 
typhoid-bacilli  in,  171 
Susceptibility  of  infected  organism,  44 


Susceptibility    to   bacterial    diseases, 

45 
Symbiosis,  41 

Sympathetic  ophthalmia,  161 
Syphilis,  314 
bacillus  of,  314 

congenital,    transmission   of,    from 
father,  319 
of,  from  mother,  320 
conveyance  of,  317 
extrauterine  infection  with,  320 
hereditary,  319 

transmission  of,  317 
heredity  of,  319 
immunity  to,  318 
intrauterine  infection  with,  320 
paternal  infection  with,  319 
portal  of  infection  for,  317 
specific  therapy  of,  318 
Syphilitic  infection,  317 


Temperature-maximum,  21 
Temperature- minimum,  21 
Temperature-optimum,  21 
Temporary  predisposition,  44 
Testicles,  typhoid-bacillus  in,  171 
Tetanus,  230 

bacillus  of,  in  soil,  391 

bacteriologic  diagnosis  of,  240 

cure  of,  241 

direct  contagion  in,  239 

idiopathic,  239 

immunity  to,  241 

in  animals,  233 

nature  of,  235 

period  of  incubation  of,  234 

portal  of  infection  for,  239 

prophylactic    inoculations   against, 

243 

rheumatic,  239 

serum- therapy  of,  242 
Tetanus-bacillus,  231 

cultural  properties  of,  232 

pure  cultures  of,  232 
Tetanus-infection   in   human    beings, 

Tetanus-toxin,  236 
Tetrad  arrangement,  21 
Tetragenus,  21 
Thallus,  336 

Therapeutic  experiments  in  tubercu- 
losis, 269 
Therapy,  specific,  72 
Thermal  death-point,  22 
Thermophilic  bacteria,  22 


440 


INDEX. 


Thiothrix,  27 

Throat,  inflammations  of,  133 

Thrush,  351 
course  of,  352 
diagnosis  of,  354 
occurrence  of,  351 
sources  of  infection  for,  353 

Thrush-deposits,  microscopic  exami- 
nation of,  351 

Thrush-fungus,    botanic  position  of, 

353 

culture  of,  353 
Tissue,  sections  of,  staining  of,  106 
Tissue-immunity,  71 
Toxalbumins,  30 
Toxic  infection,  34 
Toxins  of  anthrax-bacilli,  297 

of  cholera,  189 

specific,  29 
Toxoids,  223 
Trachoma,  161 
Traumatic  pericarditis,  150 
Trichophyton,  cultural  properties  of, 

.346 

microscopic  demonstration  of,  347 

tonsurans,  345 
Tropical  abscess  of  liver,  155 
Tubercle-bacilli,    diagnostic    demon- 
stration of,  262 

distribution  of,  260 

in  feces,  264     * 

in    fluid    from    pleural    effusions, 
264 

in  milk,  265 

in  pus,  263 

from  cold  abscesses,  264 

in  sections,  264 

in. sputum,  263 

in  urine,  264 

occurrence  of,  260 
Tubercle-bacillus,  251 

culture  of,  253 

morphology  of,  25 1 

pathogenic  activity  of,  258 

portals  of  entry  for,  257 

resistance  of,  255 

staining  of,  251 
Tuberculin,  curative  influence  of,  270 

diagnostic  utility  of,  171 

new  preparations  of,  273 

O,  273 

R,  273 

therapeutic  employment  of,  272 

treatment  of  tuberculosis  with,  270 
Tuberculosis,  251 

congenital,  268 

due  to  foreign  bodies,  276 

experimental,  in  animals,  256 

general,  260 


Tuberculosis,  general  miliary,  256 
heredity  of,  267 
localization  of,  260 
miliary,  260 
mixed  infection  in,  260 
pathologic  anatomy  of,  257 
pericarditis,  150 
pneumonia  complicating,  145 
predisposition  to,  259 
prophylaxis  of,  266 
spontaneous,  in  animals,  256 
susceptibility  to,  259 
therapeutic  experiments  in,  269 
treatment  of,  with  tuberculin,  270 
Tuberculous  cystitis,  155 

endocarditis,  149 
Typhoid  endocarditis,  149 
fever,  166 

abscess  complicating,  127 
agglutination-phenomenon       in, 

76 
bacteriologic  diagnosis  of,  174 
cure  of,  1 79 

epidemic  distribution  of,  170 
etiologic    relations    of   typhoid- 
bacilli  to,  173 
experimental     development     of, 

172 
immunity  to,  179 
infection  with,  by  way  of  diges- 
tive tract,  169 
by  way  of  lungs,  169 
through    articles    of    food, 

169 
through  drinking-water,  1 70 
mixed  infection  in,  171 
prophylaxis  of,  179 
puncture  of  spleen  in,  175 
secondary  infection  in,  171 
special  predisposition  to,  170 
temporal  and  local  predisposition 
to,  170 
intoxication,  173 

patients,    occurrence    of     typhoid- 
bacilli  in,  170 
Typhoid-bacilli     differentiated     from 
bacterium  coli  commune,  174 
dissemination  of,  169 
etiologic   relations    of,    to   typhoid 

fever,  173 
examination  of  water  for,  1 78 
in  blood,  1 71 
in  bone-marrow,  1 70 
in  feces,  171 
in  kidneys,  171 
in  liver,  170 
in  lungs,  171 
in  meninges,  171 
in  mesenteric  glands,  1 70 


INDEX. 


441 


Typhoid-bacilli  in  myocardium,  1 71 

in  Peyer's  patches,  170 

in  spleen,  170 

in  testicles,  171 

in  typhoid  patients,  170 

in  urine,  171 
Typhoid-bacillus,   appearance    of,  in 
cultures,  167 

morphology  of,  166 

portals  of  infection  for,  169 

pyogenic  activity  of,  172 

vital  activity  of,  168 
Typhoid-like  bacilli,  402 


U. 

Ulcerative  endocarditis,  149 
Urinary  phlegmon,  127 
Urine,    ammoniacal  fermentation  of, 
156 
bacteria   in,   diagnostic   and  prog- 
nostic significance  of,  158 
disinfection  of,  421 
tubercle-bacilli  in,  264 
typhoid-bacilli  in,  17 1 


V. 

Vaccination,  326 

against  anthrax,  299 
hydrophobia,  323 
Vaccinia,  326 
Varicella,  328 
Variola,  325 

nature  of  virus  of,  327 
Variolation,  326 
Vehicles,  disinfection  of,  425 
Vibrio  aquatilis  Giinther,  407 

Berolinensis,  407 

Gindha,  408 

Lissabon,  408 


Vibrio,  Massowah,  410 
Metschnikoff,  408 
phosphorescens  Dunbar,  410 
septicus,  occurrence  of,  307 

Vibrios,  17 

Virulence  of  infectious  agents,  37 

Visceral  mycoses,  350 

Vulvitis,  159 


W, 

Wash-water,  disinfection  of,  420 

Water,  bacteria  in,  401 

bacteriologic   examination  of,  389, 

396 
boiling,  disinfection  by,  41 1 
examination  of,  for  cholera-bacilli, 

195 
for  typhoid-bacilli,  178 

filtration  of,  399 

hot,  disinfection  by,  411 

method  of  examination  of,  396 

number  of  bacteria  in,  397 

of  condensation,  82 

sand-filtration  of,  399 

self- purification  of,  398 
Water-conduits,  disinfection  of,  421 
Waters,  artificial  carbonated,  401 
Water-supply,  sources  and  mode  of, 

399 
Weigert's     modification    of    Gram's 

method  of  staining,  107 
Weil's  disease,  310 
Whey,  86 

Whooping-cough,  330 
Wool-sorters   disease,  295 


Y. 

Yeast-fungi,  338 

Yeasts,  pathogenic,  341,  363 

Yellow  sarcina,  406 


CATALOGUE 

OF   THE 

MEDICAL  PUBLICATIONS 

OF 

W.  B.  SAUNDERS, 

No*   925   WALNUT   STREET,   PHILADELPHIAc 
Arranged  Alphabetically  and  Classified  under  Subjects* 


THE  books  advertised  in  this  Catalogue  as  being  sold  by  subscription  are  usually  to  be 
obtained  from  travelling  solicitors,  but  they  will  be  sent  direct  from  the  office  of  pub- 
lication (charges  of  shipment  prepaid)  upon  receipt  of  the  prices  given.     All  the  other 
books  advertised  are  commonly  for  sale  by  booksellers  in  all  parts  of  the  United  States;  but 
books  will  be  sent  to  any  address,  carriage  prepaid,  on  receipt  of  the  published  price. 

Money  may  be  sent  at  the  risk  of  the  publisher  in  either  of  the  following  ways :  A  post- 
office  money  order,  an  express  money  order,  a  bank  check,  and  in  a  registered  letter.  Money 
sent  in  any  other  way  is  at  the  risk  of  the  sender. 

See  pages  30^  Z\y  for  a  L^t  of  Contents  classified  according  to  subjects* 


LATEST  PUBLICATIONS. 


International  Text-Book  of  Surgfery.    See  page  32. 

American  Text-Book  of  Surgery — Third  (Revised)  Edition*    Seepages. 

American  Text-Book  of  Dis*  of  Eye,  Ear,  Nose,  and  Throat*    Page  3* 

American  Text-Book  of  Genito-Urinary  and  Skin  Diseases*    Page  4* 

Heisler's  Embryology*    See  page  32. 

Nancf ede's  Principles  of  Surgery*    See  page  32. 

Jackson's  Diseases  of  the  Eye*    See  page  32. 

Kyle  on  the  Nose  and  Throat*    See  page  15. 

Pryor's  Pelvic  Inflammations*    See  pages  J9  and  32. 

Abbott's  Hygiene  of  Transmissible  Diseases*    See  page  32. 

Anders'  Practice  of  Medicine — Third  (Revised)  Edition*     See  page  6* 

Vierordt's  Medical  Diagnosis — ^Fourth  (Revised)  Edition*    See  page  29. 

Church  and  Peterson's  Nervous  arid  Mental  Diseases*    See  page  8. 

Da  Gjsta's  Surgery — Revised  and  Enlarged  Edition*     See  page  JO. 

Saunders'  Medical  Hand-Atlases*    See  page  2. 

Griffith  on  the  Baby — ^Revised  Edition.     See  page  J2. 

Butler's  Materia  Medica  and  Therapeutics — Third  (Revised)  Ed*  Page  8* 

De  Schweinitz's  Diseases  of  the  Eye — Third  (Revised)  Ed.    See  page  JO. 

Vecki's  Sexual  Impotence*     See  page  28. 

Stoney's  Materia  Medica  for  Nurses.    See  page  28. 

McFarland's  Pathogenic  Bacteria — ^Revised  Edition*    See  page  J 7. 

American  Pocket  Medical  Dictionary — ^Second  (Revised)  Ed*     Page  JO. 

Stengel's  Text-Book  of  Pathology*    Second  Edition*     See  page  26. 

Hirst's  Text-Book  of  Obstetrics*    See  page  13. 


SAUNDERS'  MEDICAL  HAND-ATLASES. 


The  series  of  books  included  under  this  title  consists  of  authorized  translations  into 
English  of  the  world-famous  Lehmann  Medicinische  Handatlanten,  which  for  sci- 
entific accuracy,  pictorial  beauty,  compactness,  and  cheapness  surpass  any  similar 
volumes  ever  published.  Each  volume  contams  from  50  to  100  colored  plates,  executed 
by  the  most  skilful  German  lithographers,  besides  numerous  illustrations  in  the  text.  There 
is  a  full  and  appropriate  description  of  each  plate,  and  each  book  contains  a  condensed 
but  adequate  outline  of  the  subject  to  which  it  is  devoted. 

One  of  the  most  valuable  features  of  these  atlases  is  that  they  offer  a  ready  and  satis- 
factory substitute  for  clinical  observation.  To  those  unable  to  attend  important  clinics 
these  books  will  be  absolutely  indispensable. 

In  planning  this  series  of  books  arrangements  were  made  with  representative  publishers 
in  the  chief  medical  centers  of  the  world  for  the  publication  of  translations  of  the  atlases 
into  nine  different  languages,  the  lithographic  plates  for  all  these  editions  being  made  in  Ger- 
many, where  work  of  this  kind  has  been  brought  to  the  greatest  perfection.  The  expense  of 
making  the  plates  being  shared  by  the  various  publishers,  the  cost  to  each  one  was  materially 
reduced.  Thus  by  reason  of  their  universal  translation  and  reproduction,  the  publish- 
ers have  been  enabled  to  secure  for  these  atlases  the  best  artistic  and  professional 
talent,  to  produce  them  in  the  most  elegant  style,  and  yet  to  offer  them  at  a  pl-ice 
heretofore  unapproached  in  cheapness.  The  success  of  the  undertaking  is  demon- 
strated by  the  fact  that  the  volumes  have  already  appeared  in  nine  different  languages 
— German,  English,  French,  Italian,  Russian,  Spanish,  Danish,  Swedish-,  and  Hungarian. 

In  view  of  the  striking  success  of  these  works,  Mr.  Saunders  has  contracted  with  the 
publisher  of  the  original  German  edition  for  one  hundred  thousand  copies  of  the  atlases. 
In  consideration  of  this  enormous  undertaking,  the  publisher  has  been  enabled  to  prepare 
and  furnish  special  additional  colored  plates,  making  the  series  even  handsomer  and  more 
complete  than  was  originally  intended. 

As  an  indication  of  the  practical  value  of  the  atlases  and  of  the  favor  with  which  they 
have  been  received,  it  should  be  noted  that  the  Medical  Department  of  the  U.  S.  Army 
has  adopted  the  "Atlas  of  Operative  Surgery  "  as  its  standard,  and  has  ordered  the  book  in 
large  quantities  for  distribution  to  the  various  regiments  and  army  posts. 

The  same  careful  and  competent  editorial  supervision  has  been  secured  in  the 
English  edition  as  in  the  originals,  the  translations  being  edited  by  the  leading  American 
specialists  in  the  different  subjects. 

NOW  READY. 

Atlas  of  Internal  Medicine  and  Clinical  Diagnosis.  By  Dr.  Chr.  Jakob,  of  Erlangen.  Edited 
by  Augustus  A.  Eshner,  M.D.,  Professor  of  Clinical  Medicine  in  the  Philadelphia  Polyclinic;  At- 
tending Physician  to  the  Philadelphia  Hospital.  68  colored  plates,  and  64  illustrations  in  the  text. 
Cloth,  $3.00  net. 

Atlas  of  Legal  Medicine.  By  Dr.  E.  R.  von  Hofmann,  of  Vienna.  Edited  by  Frederick  Peter- 
son, M.D.,  Clinical  Professor  of  Mental  Diseases,  Woman's  Medical  College,  New  York  ;  Chiei 
of  Clinic,  Nervous  Dept.,  College  of  Physicians  and  Surgeons,  New  York.  With  120  colored  fig- 
ures on  56  plates,  and  193  beautiful  half-tone  illustrations.    Cloth,  53.50  net. 

Atlas  of  Diseases  of  the  Larynx.  By  Dr.  L.  GriJnw^ald,  of  Munich.  Edited  by  Charles  P. 
Grayson,  M.D.,  Lecturer  on  Laryngology  and  Rhinology  in  the  University  of  Pennsylvania; 
Physician-in-Charge,  Throat  and  Nose  Department,  Hospital  of  the  University  of  Pennsylvania. 
With  107  colored  figures  on  44  plates,  and  25  text-illustrations.    Cloth,  $2.50  net. 

Atlas  of  Operative  Surgery.  By  Dr.  O.  Zuckkrkandl,  of  Vienna.  Edited  by  J.  Chalmers 
DaCosta,  M.D.,  Clinftal  Professor  of  Surgery,  Jetterson  Medical  College,  Philadelphia  ;  Surgeon 
to  the  Philadelphia  Hospital.    With  24  colored  plates,  and  217  text  illustrations.    Cloth,  $3.00  net. 

Atlas  of  Syphilis  and  the  Venereal  Diseases.  By  Prof.  Dr.  Franz  Mracek,  of  Vienna.  Edited 
by  L.  BoLTON  Bangs,  M.  D.,  Professor  of  Genito-Urinary  Surgery,  University  and  Bellevue  Hospi- 
tal Medical  College,  New  York.  With  71  colored  plates,  16  black-and-white  illustrations,  and  122 
pages  of  text.     Cloth,  ;^3.5o  net. 

Atlas  of  External  Diseases  of  the  Eye.  By  Dr.  O.  Haab,  of  Zurich.  Edited  by  G.  E. 
DE  ScHWEiNiTZ,  M.  D.,  Profcssor  of  Ophthalmology,  Jefferson  Medical  College,  Philadelphia. 
With    76   colored    illustrations  on   40  plates,  and   228   pages   of  text.     Cloth,  ;j3.oo  net. 

Atlas  of  Skin  Diseases.  By  Prof.  Dr.  Franz  Mracek,  of  Vienna.  Edited  by  Henry  W.  Stelwagon, 
M.  D.,  Clinical  Professor  of  Dermatology,  Jefferson  Medical  College,  Philadelphia.  63  colored  plates, 
39  beautiful  half-tone  illustrations,  and  200  pages  of  text.     Cloth,  $3.50  net. 

IN  PREPARATION. 

Atlas  of  Pathological  Histology.  Atlas  of  Operative  Gynecology. 

Atlas  of  Orthopedic  Surgery.  Atlas  of  Psychiatry. 

Atlas  of  General  Surgery.  Atlas  of  Diseases  of  the  Ear. 


THE  AMERICAN  TEXT-BOOK  SERIES. 

AN  AMERICAN  TEXT-BOOK  OF  APPLIED  THERAPEUTICS. 

By  43  Distinguished  Practitioners  and  Teachers.  Edited  by  James  C. 
Wilson,  M.D.,  Professor  of  the  Practice  of  Medicine  and  of  Clinical 
Medicine  in  the  Jefferson  Medical  College,  Philadelphia.  One  hand- 
some imperial  octavo  volume  of  1326  pages.  Illustrated.  Cloth, 
II7.00  net;  Sheep  or  Half  Morocco,  $8.00  net.     Sold  by  Subscription, 

*'  As  a  work  either  for  study  or  reference  it  will  be  of  great  value  to  the  practitioner,  as 
it  is  virtually  an  exposition  of  such  clinical  therapeutics  as  experience  has  taught  to  be  oi 
the  most  value.  Taking  it  all  in  all,  no  recent  publication  on  therapeutics  can  be  compared 
with  this  one  in  practical  value  to  the  working  physician." — Chicago  Clinical  Review. 

**  The  whole  field  of  medicine  has  been  well  covered.  The  work  is  thoroughly  prac- 
tical, and  while  it  is  intended  for  practitioners  and  students,  it  is  a  better  book  for  the  general 
practitioner  than  for  the  student.  The  young  practitioner  especially  will  find  it  extremely 
suggestive  and  helpful." — The  Indian  Lancet, 

AN  AMERICAN  TEXT=BOOK  OF  THE  DISEASES  OF  CHILDREN. 
Second  Edition,  Revised. 

By  65  Eminent  Contributors.  Edited  by  Louis  Starr,  M.  D.,  Con- 
sulting Pediatrist  to  the  Maternity  Hospital,  etc.  ;  assisted  by  Thomp- 
son S.  Westcott,  M.  D.,  Attending  Physician  to  the  Dispensary 
for  Diseases  of  Children,  Hospital  of  the  University  of  Pennsyl- 
vania. In  one  handsome  imperial  octavo  volume  of  1244  pages, 
profusely  illustrated.  Cloth,  $7.00  net;  Sheep  or  Half  Morocco, 
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**  This  is  far  and  away  the  best  text-book  on  children's  diseases  ever  published  in  the 
English  language,  and  is  certainly  the  one  which  is  best  adapted  to  American  readers. 
We  congratulate  the  editor  upon  the  result  of  his  work,  and  heartily  commend  it  to  the 
attention  of  every  student  and  practitioner." — American  Journal  of  the  Medical  Sciences. 

AN  AMERICAN  TEXT-BOOK  OF  DISEASES  OF  THE  EYE,  EAR, 
NOSE,  AND  THROAT. 

By  58  Prominent  Specialists.  Edited  by  G.  E.  de  Schweinitz,  M.D, 
Professor  of  Ophthalmology  in  the  Jefferson  Medical  College,  Phila- 
delphia ;  and  B.  Alexander  Randall,  M.D.,  Professor  of  Diseases 
of  the  Ear  in  the  University  of  Pennsylvania.  Imperial  octavo,  1251 
pages;  766  illustrations,  59  of  them  in  colors.  Cloth,  ^7.00  net;  Sheep 
or  Half  Morocco,  ^8.00  net.     Sold  by  Subscription. 

litustrated  Catalogue  of  the  ^'American  Text-Books''  sent  free  upon  applicatioiu     - 


4  Medical  Pahlications  of  W.  B,  Saunders. 

AN  AMERICAN  TEXT-BOOK   OF   GENITO-URINARY  AND  SKIN 
DISEASES. 

By  47  Eminent  Specialists  and  Teachers.  Edited  by  L.  Bolton 
Bangs,  M.  D.,  Professor  of  Genito- Urinary  Surgery,  University  and 
Bellevue  Hospital  Medical  College,  New  York  ;  and  W.  A.  Hard- 
AWAY,  M.  D.,  Professor  of  Diseases  of  the  Skin,  Missouri  Medical 
College.  Imperial  octavo  volume  of  1229  pages,  with  300  engravings 
and  20  full-page  colored  plates.  Cloth,  ^7.00  net;  Sheep  or  Half 
Morocco,  ;^8.oo  net.     So/d  by  SubscHption. 

"This  volume  is  one  of  the  best  yet  issued  of  the  publisher's  series  of '  American  Text- 
Books.'  The  list  of  contributors  represents  an  extraordinary  array  of  talent  and  extended 
experience.  The  book  will  easily  take  the  place  in  comprehensiveness  and  value  of  the 
half  dozen  or  more  costly  works  on  these  subjects  which  have  heretofore  been  necessary  to 
a  well-equipped  library." — Neiv  York  Polyclinic. 

AN  AMERICAN  TEXT=BOOK  OF  GYNECOLOGY,  MEDICAL  AND 
SURGICAL.     Second  Edition,  Revised. 

By  10  of  the  Leading  Gynecologists  of  America.  Edited  by  J-  M. 
Baldy,  M.  D.,  Professor  of  Gynecology  in  the  Philadelphia  Polyclinic, 
etc.  Handsome  imperial  octavo  volume  of  718  pages,  with  341  illus- 
trations in  the  text,  and  38  colored  and  half-tone  plates.  Cloth,  $6.00 
net ;  Sheep  or  Half  Morocco,  ^7.00  net.     Sold  by  Subscription. 

"  It  is  practical  from  beginning  to  end.  Its  descriptions  of  conditions,  its  recommen- 
dations for  treatment,  and  above  all  the  necessary  technique  of  different  operations,  are 
clearly  and  admirably  presented.  .  .  .  It  is  well  up  to  the  most  advanced  views  of  the 
day,  and  embodies  all  the  essential  points  of  advanced  American  gynecology.  It  is  destined 
to  make  and  hold  a  place  in  gynecological  literature  which  will  be  peculiarly  its  own."— 
Medical  Record,  New  York. 

AN  AMERICAN  TEXT-BOOK  OF  LEGAL  MEDICINE  AND  TOXI- 
COLOGY. 

Edited  by  Frederick  Peterson,  M.D.,  Clinical  Professor  of  Mental 
Diseases  in  the  Woman's  Medical  College,  New  York;  Chief  of  Clinic, 
Nervous  Department,  College  of  Physicians  and  Surgeons,  New  York ; 
and  Walter  S.  Haines,  M.D.,  Professor  of  Chemistry,  Pharmacy, 
and  Toxicology  in  Rush  Medical  College,  Chicago.    In  Preparation, 

AN  AMERICAN  TEXT=BOOK  OF  OBSTETRICS. 

By  15  Eminent  American  Obstetricians.  Edited  by  Richard  C.  Nor- 
Ris,  M.D.;  Art  Editor,  Robert  L.  Dickinson,  M.D.  One  handsome 
imperial  octavo  volume  of  1014  pages,  with  nearly  900  beautiful  colored 
and  half-tone  illustrations.  Cloth,  ^7.00  netj  Sheep  or  Half  Morocco, 
$8.00  net.     Sold  by  Subscription. 

"  Permit  me  to  say  that  your  American  Text-Book  of  Obstetrics  is  the  most  magnificent 
medical  work  that  I  have  ever  seen.  I  congratulate  you  and  thank  you  for  this  superb  work, 
which  alone  is  sufficient  to  place  you  first  in  the  ranks  of  medical  publishers." — Alexander 
J.  C.  Skene,  Professor  of  Gynecology  in  the  Long  Island  College  Hospital,  Brooklyn,  N.  Y. 

"  This  is  the  most  sumptuously  illustrated  work  on  midwifery  that  has  yet  appeared.  In 
the  number,  the  excellence,  and  the  beauty  of  production  of  the  illustrations  it  far  surpasses 
every  other  book  upon  the  subject.  This  feature  alone  makes  it  a  work  which  no  medical 
library  should  omit  to  purchase." — British  Medical  Journal. 

'*  As  an  authority,  as  a  book  of  reference,  as  a  '  working  book  '  for  the  student  or  prac- 
titioner, we  commend  it  because  we  believe  there  is  no  better." — American  Journal  of  the 
Medical  Sciences. 

Illustrated  Catalogue  of  the  ** American  Text-Books  '*  sent  free  ttpon  application* 


Medical  Publications  of  W.  B.  Saunders.  5 

AN  AMERICAN  TEXT-BOOK  OF  PATHOLOGY. 

Edited  by  John  Guiteras,  M.D.,  Professor  of  General  Pathology  and 
of  Morbid  Anatomy  in  the  University  of  Pennsylvania;  and  David 
RiESMAN,  M.D.,  Demonstrator  of  Pathological  Histology  in  the 
University  of  Pennsylvania.     In  Preparation. 

AN  AMERICAN  TEXT-BOOK  OF  PHYSIOLOGY. 

By  I  o  of  the  Leading  Physiologists  of  America.  Edited  by  William 
H.  Howell,  Ph.D.,  M.D.,  Professor  of  Physiology  in  the  Johns  Hop- 
kins University,  Baltimore,  Md.  One  handsome  imperial  octavo 
volume  of  1052  pages.  Illustrated.  Cloth,  $6.00  net;  Sheep  or  Half 
Morocco,  $7.00  net.     Sold  by  Subscription. 

"  We  can  commend  it  most  heartily,  not  only  to  all  students  of  physiology,  but  to  every 
physician  and  pathologist,  as  a  valuable  and  comprehensive  work  of  reference,  written  by 
men  who  are  of  eminent  authority  in  their  own  special  subjects." — London  Lancet. 

**  To  the  practitioner  of  medicine  and  to  the  advanced  student  this  volume  constitutes, 
we  believe,  the  best  exposition  of  the  present  status  of  the  science  of  physiology  in  the 
English  language." — American  Journal  of  the  Medical  Sciences. 

AN   AMERICAN  TEXT-BOOK  OF  SURGERY.    Third  Edition. 

By  II  Eminent  Professors  of  Surgery.  Edited  by  William  W.  Keen, 
M.D.,  LL.D.,  and  J.  William  White,  M.D.,  Ph.D.  Handsome  im- 
perial octavo  volume  of  1230  pages,  with  496  wood-cuts  in  the  text, 
and  37  colored  and  half-tone  plates.  Thoroughly  revised  and  enlarged, 
with  a  section  devoted  to  ''  The  Use  of  the  Rontgen  Rays  in  Surgery." 
Cloth,  $7.00  net;  Sheep  or  Half  Morocco,  ^8.00  net. 

*•  Personally,  I  should  not  mind  it  being  called  THE  Text-Book  (instead  of  A  Text- 
Book)  ,  for  I  know  of  no  single  volume  which  contains  so  readable  and  complete  an  account 
of  the  science  and  art  of  Surgery  as  this  does." — Edmund  Owen,  F.R.C.S.,  Member  of 
the  Board  of  Examiners  of  the  Royal  College  of  Surgeons,  England. 

*'  If  this  text-book  is  a  fair  reflex  of  the  present  position  of  American  surgery,  we  must 
admit  it  is  of  a  very  high  order  of  merit,  and  that  English  surgeons  will  have  to  look  very 
carefully  to  their  laurels  if  they  are  to  preserve  a  position  in  the  van  of  surgical  practice." — 
London  Lancet. 

AN  AMERICAN  TEXT-BOOK  OF  THE  THEORY  AND  PRACTICE 
OF  MEDICINE. 

By  12  Distinguished  American  Practitioners.  Edited  by  William 
Pepper,  M.D.,  LL.D.,  Professor  of  the  Theory  and  Practice  of  Medi- 
cine and  of  Clinical  Medicine  in  the  University  of  Pennsylvania.  Two 
handsome  imperial  octavo  volumes  of  about  1000  pages  each.  Illus- 
trated. Prices  per  volume :  Cloth,  ^5.00  net ;  Sheep  or  Half  Morocco, 
$6.00  net.     Sold  by  Subscription. 

"  I  am  quite  sure  it  will  commend  itself  both  to  practitioners  and  students  of  medicine, 
and  become  one  of  our  most  popular  text-books." — Alfred  Loomis,  M.D.,  LL.D.,  Pro- 
fessor  of  Pathology  and  Practice  of  Medicine,  University  of  the  City  of  New  York. 

•*  We  reviewed  the  first  volume  of  this  work,  and  said  :  *  It  is  undoubtedly  one  of  the 
best  text-books  on  the  practice  of  medicine  which  we  possess.'  A  consideration  of  the 
second  and  last  volume  leads  us  to  modify  that  verdict  and  to  say  that  the  completed  work 
is  in  our  opinion  the  best  of  its  kind  it  has  ever  been  our  fortune  to  see. " — New  York  Medical 
JourncU. 

lUtsstrated  Catalos^e  of  the  ** American  Text-Books''  sent  free  tfpon  application* 


6  Medical  Publications  of  W,  B,  Saunders. 

AN  AMERICAN  YEAR-BOOK  OF  MEDICINE  AND  SURGERY. 

A  Yearly  Digest  of  Scientific  Progress  and  Authoritative  Opinion  in  all 
branches  of  Medicine  and  Surgery,  drawn  from  journals,  monographs, 
and  text-books  of  the  leading  American  and  Foreign  authors  and 
investigators.  Collected  and  arranged,  with  critical  editorial  com- 
ments, by  eminent  American  specialists  and  teachers,  under  the  general 
editorial  charge  of  George  M.  Gould,  M.D.  One  handsome  imperial 
octavo  volume  of  about  1200  pages.  Uniform  in  style,  size,  and 
general  make-up  with  the  ** American  Text-Book"  Series.  Cloth, 
$6.50  net;  Half  Morocco,  $7.50  net.     Sold  by  Subscription. 

**  It  is  difficult  to  know  which  to  admire  most — the  research  and  industry  of  the  distin- 
guished band  of  experts  whom  Dr.  Gould  has  enlisted  in  the  service  of  the  Year-Book,  or  the 
wealth  and  abundance  of  the  contributions  to  every  department  of  science  that  have  been 
deemed  worthy  of  analysis.  .  .  .  It  is  much  more  than  a  mere  compilation  of  abstracts, 
for,  as  each  section  is  entrusted  to  experienced  and  able  contributors,  the  reader  has  the 
advantage  of  certain  critical  commentaries  and  expositions  .  .  .  proceeding  from  writers 
fully  qualified  to  perform  these  tasks.  .  .  .  It  is  emphatically  a  book  which  should  find 
a  place  in  every  medical  library,  and  is  in  several  respects  more  useful  than  the  famous 
*Jahrbucher'  of  Germany." — London  Lancet. 

THE  AMERICAN  POCKET  MEDICAL  DICTIONARY. 

[See  D  or  I  and' s  Pocket  Dictionary  ^  page  10.] 

ANDERS'  PRACTICE  OF  MEDICINE.  Third  Revised  Edition. 
A  Text-Book  of  the  Practice  of  Medicine.  By  James  M.  Anders, 
M.D.,  Ph.D.,  LL.D.,  Professor  of  the  Practice  of  Medicine  and  of 
.  Clinical  Medicine,  Medico- Chirurgical  College,  Philadelphia.  In  one 
handsome  octavo  volume  of  1292  pages,  fully  illustrated.  Cloth, 
$5.50  net;  Sheep  or  Half  Morocco,  ^^6.50  net. 

"It  is  an  excellent  book, — concise,  comprehensive,  thorough,  and  up  to  date.  It  is  a 
credit  to  you  ;  but,  more  than  that,  it  is  a  credit  to  the  profession  of  Philadelphia — to  us." 
James  C.  Wilson,  Professor  of  the  Practice  of  Medicine  and  Clinical  Medicine^  Jefferson 
Medical  College ^  Philadelphia. 

ASHTON'S  OBSTETRICS.     Fourth  Edition,  Revised. 

Essentials  of  Obstetrics.  By  W.  Easterly  Ashton,  M.D.,  Pro* 
fessor  of  Gynecology  in  the  Medico-Chirurgical  College,  Philadelphia. 
Crown  octavo,  252  pages;  75  illustrations.  Cloth,  ^1.09;  interleaved 
for  notes,  ^1.25. 

[See  Saunders*  Question- Compends,  page  21.] 

"  Embodies  the  vphole  subject  in  a  nut-shell.  We  cordially  recommend  it  to  our  read- 
ers."— Chicago  Medical  Times. 

BALL*S  BACTERIOLOGY.     Third  Edition,  Revised. 

Essentials  of  Bacteriology  ;  a  Concise  and  Systematic  Introduction 
to  the  Study  of  Micro-organisms.  By  M.  V.  Ball,  M.D.,  Bacteriol- 
ogist to  St.  Agnes'  Hospital,  Philadelphia,  etc.  Crown  octavo,  218 
pages;  82  illustrations,  some  in  colors,  and  5  plates.  Cloth,  $1.00; 
interleaved  for  notes,  $1.25. 

[See  Saunders*  Question- Compends,  page  21.] 

"  The  student  or  practitioner  can  readily  obtain  a  knowledge  of  the  subject  from  a  perusal 
of  this  book.     The  illustrations  are  clear  and  satisfactory." — Medical  Record,  New  York. 


Medical  Publications  of  W.  B.  Saunders,  1 

BASTINGS  BOTANY. 

Laboratory  Exercises  in  Botany.  By  Edson  S.  Bastin,  M.A., 
late  Professor  of  Materia  Medica  and  Botany,  Philadelphia  College  of 
Pharmacy.    Octavo  volume  of  536  pages,  with  87  plates.    Cloth,  ^^2.50. 

**It  is  unquestionably  the  best  text-book  on  the  subject  that  has  yet  appeared.  The 
work  is  eminently  a  practical  one.  We  regard  the  issuance  of  this  book  as  an  important 
event  in  the  history  of  pharmaceutical  teaching  in  this  country,  and  predict  for  it  an  unquali- 
fied success." — Alumni  Report  to  the  Philadelphia  College  of  Pharmacy. 

"There  is  no  work  like  it  in  the  pharmaceutical  or  botanical  literature  of  this  country, 
and  we  predict  for  it  a  wide  circulation." — American  Journal  of  Pharmacy, 

BECK'S  SURGICAL  ASEPSIS. 

A  Manual  of  Surgical  Asepsis.  By  Carl  Beck,  M.Do,  Surgeon  to 
St.  Mark's  Hospital  and  the  New  York  German  Poliklinik,  etc.  306 
pages;  65  text-illustrations,  and  12  full-page  plates.     Cloth,  ^1.25  net. 

"An  excellent  exposition  of  the  *very  latest'  in  the  treatment  of  wounds  as  practised 
by  leading  German  and  American  surgeons." — Birmingham  (Eng.)  Medical  Review, 

"This  little  volume  can  be  recommended  to  any  who  are  desirous  of  learning  the  details 
of  asepsis  in  surgery,  for  it  will  serve  as  a  trustworthy  guide." — London  Lancet. 

BOISLINIERE'S  OBSTETRIC  ACCIDENTS,  EMERGENCIES,  AND 
OPERATIONS. 
Obstetric  Accidents,  Emergencies,  and  Operations.     By  L.  Ch. 

BoiSLiNiERE,  M.D.,  late  Emeritus  Professor  of  Obstetrics,  St.  Louis 
Medical  College.    381  pages,  handsomely  illustrated.    Cloth,  ;^2.oo  net. 

**  It  is  clearly  and  concisely  written,  and  is  evidently  the  work  of  a  teacher  and  practi- 
tioner of  large  experience." — British  Medical  Journal. 

"  A  manual  so  useful  to  the  student  or  the  general  practitioner  has  not  been  brought  to 
our  notice  in  a  long  time.  The  field  embraced  in  the  title  is  covered  in  a  terse,  interesting 
way." —  Yale  Medical  Journal. 

BROCKWAY'S  MEDICAL  PHYSICS.     Second  Edition,  Revised. 
Essentials  of  Medical  Physics.     By  Fred  J.  Brockway,  M.D., 
Assistant  Demonstrator  of  Anatomy  in  the  College  of  Physicians  and 
Surgeons,  New  York.     Crown  octavo,  330  pages;   155  fine  illustrations. 
Cloth,  ^i.oo  net ;  interleaved  for  notes,  ^1.25  net. 

[See  Saunders*  Que stion-Comp ends,  page  21.] 

"  The  student  who  is  well  versed  in  these  pages  will  certainly  prove  qualified  to  com. 
prehend  with  ease  and  pleasure  the  great  majority  of  questions  involving  physical  principles 
likely  to  be  met  with  in  his  medical  studies." — American  Practitioner  and  News. 

**We  know  of  no  manual  that  affords  the  medical  student  a  better  or  more  concise 
exposition  of  physics,  and  the  book  may  be  commended  as  a  most  satisfactory  presentation 
of  those  essentials  that  are  requisite  in  a  course  in  medicine." — New  York  Medical  JoumaU 

**  It  contains  all  that  one  need  know  on  the  subject,  is  well  written,  and  is  copiously 
illustrated." — Medical  Record^  New  York. 

BURR  ON  NERVOUS  DISEASES. 

A  Manual  of  Nervous  Diseases.  By  Charles  W.  Burr,  M.D., 
Clinical  Professor  of  Nervous  Diseases,  Medico-Chirurgical  College, 
Philadelphia;  Pathologist  to  the  Orthopedic  Hospital  and  Infirmary 
for  Nervous  Diseases;  Visiting  Physician  to  St.  Joseph's  Hospital,  etc. 
In  Preparation. 


8  Medical  Publications  of  W.  B.  Saunders. 

BUTLER'S  MATERIA  MEDICA,  THERAPEUTICS,  AND  PHAR- 
MACOLOGY. Third  Edition,  Revised. 
A  Text-Book  of  Materia  Medica,  Therapeutics,  and  Pharma- 
coJog^y.  By  George  F.  Butler,  Ph.G.,  M.D.,  Professor  of  Materia 
Medica  and  of  Clinical  Medicine  in  the  College  of  Physicians  and 
Surgeons,  Chicago ;  Professor  of  Materia  Medica  and  Therapeutics, 
Northwestern  University,  Woman's  Medical  School,  etc.  Octavo,  874 
pages,  illustrated.     Cloth,  ^^4.00  net;    Sheep,  ^5.00  net. 

**  Taken  as  a  whole,  the  book  may  fairly  be  considered  as  one  of  the  most  satisfactory 
of  any  single-volume  works  on  materia  medica  in  the  market," — Journal  of  the  American 
Medical  Association. 

CERNA  ON  THE  NEWER  REMEDIES.  Second  Edition,  Revised. 
Notes  on  the  Newer  Remedies,  their  Therapeutic  Applications 
and  Modes  of  Administration.  By  David  Cerna,  M.D.,  Ph.D., 
formerly  Demonstrator  of  and  Lecturer  on  Experimental  Therapeutics 
in  the  University  of  Pennsylvania ;  Demonstrator  of  Physiology  in  the 
Medical  Department  of  the  University  of  Texas.  Rewritten  and 
greatly  enlarged.     Post-octavo,   253  pages.     Cloth,  ^1.25. 

"  The  appearance  of  this  new  edition  of  Dr.  Cerna's  very  valuable  work  shows  that  it 
is  properly  appreciated.  The  book  ought  to  be  in  the  possession  of  every  practising  physi- 
cian."—iV^  York  Medical  Journal. 

CHAPIN  ON  INSANITY. 

A  Compendium  of  Insanity.     By  John  B.  Chapin,  M.D.,  LL.D., 

Physician-in-Chief,  Pennsylvania  Hospital  for  the  Insane ;  late  Physi- 
cian-Superintendent of  the  Willard  State  Hospital,  New  York ;  Hon- 
orary Member  of  the  Medico-Psychological  Society  of  Great  Britain, 
of  the  Society  of  Mental  Medicine  of  Belgium.  i2mo,  234  pages, 
illustrated.     Cloth,  $1.25  net. 

"  The  prafctical  parts  of  Dr.  Chapin's  book  are  what  constitute  its  distinctive  merit.  We 
desire  especially  to  call  attention  to  the  fact  that  on  the  subject  of  therapeutics  of  insanity 
the  work  is  exceedingly  valuable.  It  is  not  a  made  book,  but  a  genuine  condensed  thesis, 
which  has  all  the  value  of  ripe  opinion  and  all  the  charm  of  a  vigorous  and  natural  style." — 
Philadelphia  Medical  Journal. 

CHAPMAN'S  MEDICAL  JURISPRUDENCE  AND  TOXICOLOGY. 
Second  Edition,  Revised. 
Medical  Jurisprudence  and  Toxicology.  By  Henry  C.  Chapman, 
M.D.,  Professor  of  Institutes  of  Medicine  and  Medical  Jurisprudence 
in  the  Jefferson  Medical  College  of  Philadelphia.  254  pages,  with  55 
illustrations  and  3  full-page  plates  in  colors.     Cloth,  ^1.50  net. 

"The  best  book  of  its  class  for  the  undergraduate  that  we  know  of." — New  York 
Medical  Times. 

CHURCH  AND  PETERSON'S  NERVOUS  AND  MENTAL  DISEASES. 
Nervous  and  Mental  Diseases.  By  Archibald  Church,  M.  D., 
Professor  of  Mental  Diseases  and  Medical  Jurisprudence  in  the  North- 
western University  Medical  School,  Chicago ;  and  Frederick  Peter- 
son, M.  D.,  Clinical  Professor  of  Mental  Diseases,  Woman's  Medical 
College,  N.  Y.;  Chief  of  Clinic,  Nervous  Dept.,  College  of  Physi- 
cians and  Surgeons,  N.  Y.  Handsome  octavo  volume  of  843  pages, 
profusely  illustrated.     Cloth,  ;^5.oo  net;  Half  Morocco,  ;^6.oo  net. 


Medical  Publications  of  W.  B.  Saunders.  9 

CLARKSON'S  HISTOLOGY. 

A   Text-Book   of    Histology,    Descriptive   and    Practical.      By 

Arthur  Clarkson,  M.B.,  CM.  Edin.,  formerly  Demonstrator  of 
Physiology  in  the  Owen's  College,  Manchester;  late  Demonstrator  of 
Physiology  in  Yorkshire  College,  Leeds.  Large  octavo,  554  pages; 
22  engravings  in  the  text,  and  174  beautifully  colored  original  illustra- 
tions.     Cloth,  strongly  bound,  ^4.00  net. 

"  The  work  must  be  considered  a  valuable  addition  to  the  list  of  available  text- books, 
and  is  to  be  highly  recommended." — JVeza  York  Medical  Journal. 

"This  is  one  of  the  best  works  for  students  we  have  ever  noticed.  We  predict  that  the 
book  will  attain  a  well-deserved  popularity  among  our  students." — Chicago  Medical  Recorder. 

CLIMATOLOGY. 

Transactions  of  the  Eighth  Annual  Meeting  of  the  American 
Climatological  Association,  held  in  Washington,  September  22-25, 
1891.  Forming  a  handsome  octavo  volume  of  276  pages,  uniform  with 
remainder  of  series.      (A  limited  quantity  only.)     Cloth,  ^1.50. 

COHEN  AND  ESHNER'S  DIAGNOSIS. 

Essentials  of  Diagnosis.  By  Solomon  Solis-Cohen,  M.D.,  Pro- 
fessor of  Clinical  Medicine  and  Applied  Therapeutics  in  the  Philadel- 
phia Polyclinic  ;  and  Augustus  A.  Eshner,  M.D.,  Professor  of  Clinical 
Medicine  in  the  Philadelphia  Polyclinic.  Post-octavo,  382  pages;  55 
illustrations.     Cloth,  $1.50  net. 

[See  Saunders'  Question- Compends,  page  21.] 

**We  can  heartily  commend  the  book  to  all  those  who  contemplate  purchasing  a  'com- 
pend.'  It  is  modern  and  complete,  and  will  give  more  satisfaction  than  many  other  works 
which  are  perhaps  too  prolix  as  well  as  behind  the  times." — Medical  Review,  St.  Louis. 

CORWIN'S  PHYSICAL  DIAGNOSIS.     Third  Edition,  Revised. 

Essentials  of  Physical  Diagnosis  of  the  Thorax.  By  Arthur 
M,  CoRWiN,  A.M.,  M.D.,  Demonstrator  of  Physical  Diagnosis  in  Rush 
Medical  College,  Chicago ;  Attending  Physician  to  Central  Free  Dis- 
pensary, Department  of  Rhinology,  Laryngology,  and  Diseases  of  the 
Chest,  Chicago.   219  pages,  illustrated.   Cloth,  flexible  covers,  $1.25  net. 

**  It  is  excellent.  The  student  who  shall  use  it  as  his  guide  to  the  careful  study  of 
physical  exploration  upon  normal  and  abnormal  subjects  can  scarcely  fail  to  acquire  a  good 
working  knowledge  of  the  subject." — Philadelphia  Polyclinic. 

'*A  most  excellent  little  work.  It  brightens  the  memory  of  the  differential  diagnostic 
signs,  and  it  arranges  orderly  and  in  sequence  the  various  objective  phenomena  to  logical 
solution  of  a  careful  diagnosis. ' ' — Journal  of  Nervous  and  Mental  Diseases. 

CRAGIN'S  GYNAECOLOGY.     Fourth  Edition,  Revised. 

Essentials  of  Gynaecology.  By  Edwin  B.  Cragin,  M.  D.,  Lecturer 
in  Obstetrics,  College  of  Physicians  and  Surgeons,  New  York.  Crown 
octavo,  200  pages;  62  illustrations.  Cloth,  ^i.oo  ;  interleaved  for  notes, 
$1.25. 

\^QQ  Saunders'  Question- Compends,  page  21.  J 

"A  handy  volume,  and  a  distinct  improvement  on  students'  compends  in  general.  No 
author  who  was  not  himself  a  practical  gynecologist  could  have  consulted  the  student's  needs 
so  thoroughly  as  Dr.  Cragin  has  done," — Medical  Record,  New  York. 


12  Medical  Publications  of  W,  B,  Saunders. 

GRAFSTROM'S   MECHANO=THERAPY. 

A  Text=Book  of  Mechano-Therapy  (Massage  and  Medical  Gym- 
nastics). By  Axel  V.  Grafstrom,  B.  Sc,  M.  D.,  late  Lieutenant  in 
the  Royal  Swedish  Army ;  late  House  Physician  City  Hospital,  Black- 
well's  Island,  New  York.    i2mo,  139  pages,  illustrated.    Cloth,  ^i.oo  net. 

GRIFFITH  ON  THE  BABY.     Second  Edition,  Revised. 

The  Care  of  the  Baby.  By  J.  P.  Crozer  Griffith,  M.D.,  Clini- 
cal Professor  of  Diseases  of  Children,  University  of  Pennsylvania; 
Physician  to  the  Children's  Hospital,  Philadelphia,  etc.  i2mo,  404 
pages,  with  67  illustrations  in  the  text,  and  5  plates.     Cloth,  ;^i.5o. 

"  The  best  book  for  the  use  of  the  young  mother  with  which  we  are  acquainted.  .  .  . 
There  are  vpry  few  general  practitioners  who  could  not  read  the  book  through  with  advan- 
tage. ' ' — Archives  of  Pediatrics. 

"The  whole  book  is  characterized  by  rare  good  sense,  and  is  evidently  written  by  a 
master  hand.  It  can  be  read  with  benefit  not  only  by  mothers  but  by  medical  students  and 
by  any  practitioners  who  have  not  had  large  opportunities  for  observing  children." — Ameri- 
can Jourtial  of  Obstetrics. 

GRIFFITH'S  WEIGHT  CHART. 

Infant's  Weight  Chart.  Designed  by  J.  P.  Crozer  Griffith,  M.  D.  , 
Clinical  Professor  of  Diseases  of  Children  in  the  University  of  Penn- 
sylvania, etc.      25  charts  in  each  pad.     Per  pad,  50  cents  net. 

A  convenient  blank  for  keeping  a  record  of  the  child's  weight  during  the  first  two  years 
of  life.  Printed  on  each  chart  is  a  curve  representing  the  average  weight  of  a  healthy  infant, 
so  that  any  deviation  from  the  normal  can  readily  be  detected. 

GROSS,  SAMUEL  D.,  AUTOBIOGRAPHY  OF. 

Autobiography  of  Samuel  D.  Gross,  M.D.,  Emeritus  Professor  of 
Surgery  in  the  Jefferson  Medical  College,  Philadelphia,  with  Remi- 
niscences of  His  Times  and  Contemporaries.  Edited  by  his  Sons, 
Samuel  W.  (jROSS,  M.D.,  LL.D.,  late  Professor  of  Principles  of  Sur- 
gery and  of  Clinical  Surgery  in  the  Jefferson  Medical  College,  and 
A.  Haller  Gross,  A.M.,  of  the  Philadelphia  Bar.  Preceded  by  a 
Memoir  of  Dr.  Gross,  by  the  late  Austin  Flint,  M.D.,  LL.D.  In 
two  handsome  volumes,  each  containing  over  400  pages,  demy  octavo, 
extra  cloth,  gilt  tops,  with  fine  Frontispiece  engraved  on  steel.  Price 
per  volume,  ^2.50  net. 

**  Dr.  Gross  was  perhaps  the  most  eminent  exponent  of  medical  science  that  America 
has  yet  produced.  His  Autobiography,  related  as  it  is  with  a  fulness  and  completeness 
seldom  to  be  found  in  such  works,  is  an  interesting  and  valuable  book.  He  comments  on 
many  things,  especially,  of  course,  on  medical  men  and  medical  practice,  in  a  very  interest- 
ing way," — The  Spectator,  London,  England. 

HAMPTON'S  NURSING.  Second  Edition,  Revised  and  Enlarged. 
Nursing :  Its  Principles  and  Practice.  By  Isabel  Adams  Hamp- 
ton, Graduate  of  the  New  York  Training  School  for  Nurses  attached 
to  Bellevue  Hospital ;  late  Superintendent  of  Nurses  and  Principal  of 
the  Training  School  for  Nurses,  Johns  Hopkins  Hospital,  Baltimore, 
Md.    12  mo,  512  pages,  illustrated.     Cloth,  ^2.00  net. 

"  Seldom  have  we  perused  a  book  upon  the  subject  that  has  given  us  so  much  pleasure 
as  the  one  before  us.  We  would  strongly  urge  upon  the  members  of  our  own  profession  the 
need  of  a  book  like  this,  for  it  will  enable  each  of  us  to  become  a  training  school  in  him- 
self."— Ontario  Medical  Journal. 


Medical  Publications  of  W.  B,  Saunders.  13 


HARE'S  PHYSIOLOGY.  Fourth  Edition,  Revised. 

Essentials  of  Physiology.  By  H.  A.  Hare,  M.D.,  Professor  of 
Therapeutics  and  Materia  Medica  in  the  Jefferson  Medical  College  of 
Philadelphia.  Crown  octavo,  239  pages.  Cloth,  ^i.oo  net;  inter- 
leaved for  notes,  ^^1.25  net. 

[See  Saunders*  Question- Compends,  page  21.] 

"  The  best  condensation  of  physiological  knowledge  we  have  yet  seen." — Medical 
Record,  New  York. 

HARrS  DIET  IN  SICKNESS  AND  IN  HEALTH. 

Diet  in  Sickness  and  in  Health.  By  Mrs.  Ernest  Hart,  formerly 
Student  of  the  Faculty  of  Medicine  of  Paris  and  of  the  London  School 
of  Medicine  for  Women ;  with  an  Introduction  by  Sir  Henry 
Thompson,  F.R.C.S.,  M.D.,  London.     220  pages..      Cloth,  ^1.50. 

"  We  recommend  it  cordially  to  the  attention  of  all  practitioners ;  both  to  them  and  lo 
their  patients  it  may  be  of  the  greatest  service." — New  York  Medical  Journal. 

HAYNES'  ANATOMY. 

A  Manual  of  Anatomy.  By  Irving  S.  Haynes,  M.D.,  Adjunct 
Professor  of  Anatomy  and  Demonstrator  of  Anatomy,  Medical  Depart- 
ment of  the  New  York  University,  etc.  680  pages,  illustrated  with  42 
diagrams  in  the  text,  and  134  full-page  half-tone  illustrations  from 
original  photographs  of  the  author's  dissections.      Cloth,  ^^2.50  net. 

"  This  book  is  the  work  of  a  practical  instructor — one  who  knows  by  experience  the 
requirements  of  the  average  student,  and  is  able  to  meet  these  requirements  in  a  very  satis- 
factory way.     The  book  is  one  that  can  be  commended." — Medical  Record,  New  York. 

HEISLER'S  EMBRYOLOGY. 

A  Text=Book  of  Embryology.  By  John  C.  Heisler,  M.D.,  Pro- 
fessor of  Anatomy  in  the  Medico- Chirurgical  College,  Philadelphia.  Oc- 
tavo volume  of  405  pages,  handsomely  illustrated.    Cloth,  $2.50  net. 

HIRST'S  OBSTETRICS. 

A  Text=Book  of  Obstetrics.  By  Barton  Cooke  Hirst,  M.  D., 
Professor  of  Obstetrics  in  the  University  of  Pennsylvania.  Handsome 
octavo  volume  of  848  pages,  with  618  illustrations,  and  7  colored 
plates.     Cloth,  ^5.00  net;   Sheep  or  Half  Morocco,  ;^6.oo  net. 

"The  illustrations  are  numerous  and  are  works  of  art,  many  of  them  appearing  for  the 
first  time.  The  arrangement  of  the  subject-matter,  the  foot-notes,  and  index  are  beyond 
criticism.  As  a  true  model  of  what  a  modern  text-book  on  obstetrics  should  be,  we  feel 
justified  in  affirming  that  Dr.  Hirst's  book  is  without  a  rival." — New  York  Medical  Record. 

HYDE  AND  MONTGOMERY  ON  SYPHILIS  AND  THE  VENEREAL 
DISEASES. 
Syphilis  and  the  Venereal   Diseases.     By  James  Nevins   Hyde, 

M.D.,  Professor  of  Skin  and  Venereal  Diseases,  and  Frank  H.  Mont- 
gomery, M.D.,  Lecturer  on  Dermatology  and  Genito-Urinary  Diseases 
in  Rush  Medical  College,  Chicago,  111.  618  pages,  profusely  illustrated. 
Cloth,  $2.50  net. 

•*  We  can  commend  this  manual  to  the  student  as  a  help  to  him  in  his  study  of  venereal 
diseases. ' ' — Liverpool  Medico-  Chirurgical  Journal. 

"The  best  student's  manual  which  has  appeared  on  the  subject." — SL  Louis  Medical 
and  Surgical  Journal. 


14  Medical  Publications  of  W,  B,  Saunders, 

JACKSON  AND  GLEASON'S  DISEASES  OF  THE  EYE,  NOSE,  AND 
THROAT.  Second  Edition,  Revised. 
Essentials  of  Refraction  and  Diseases  of  the  Eye.  By  Edward 
Jackson,  A.M.,  M.D.,  Professor  of  Diseases  of  the  Eye  in  the  Phila- 
delphia Polyclinic  and  College  for  Graduates  in  Medicine ;  and — 
Essentials  of  Diseases  of  the  Nose  and  Throat.  By  E.  Bald- 
win Gleason,  M.D.  ,  Surgeon-in-Charge  of  the  Nose,  Throat,  and 
Ear  Department  of  the  Northern  Dispensary  of  Philadelphia.  Two 
volumes  in  one.  Crown  octavo,  290  pages;  124  illustrations.  Cloth, 
^i.oo;  interleaved  for  notes,  ^1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"  Of  great  value  to  the  beginner  in  these  branches.  The  authors  are  both  capable  men, 
and  know  what  a  student  most  needs." — Medical  Record,  New  York. 

KEATING'S  DICTIONARY.     Second  Edition,  Revised. 

A  New  Pronouncing  Dictionary  of  Medicine,  with  Phonetic 
Pronunciation,  Accentuation,  Etymology,  etc.  By  John  M. 
Keating,  M.D.,  LL.D.,  Fellow  of  the  College  of  Physicians  of  Phila- 
delphia; Vice-President  of  the  American  Paediatric  Society;  Editor 
"Cyclopaedia  of  the  Diseases  of  Children,"  etc.;  and  Henry 
Hamilton,  Author  of  *•' A  New  Translation  of  Virgil's  ^neid  into 
English  Rhyme,"  etc.;  with  the  collaboration  of  J.  Chalmers  Da- 
Costa,  M.D.,  and  Frederick  A.  Packard,  M.D.  With  an  Appendix 
containing  Tables  of  Bacilli,  Micrococci,  Leucoma'ines,  Ptomaines; 
Drugs  and  Materials  used  in  Antiseptic  Surgery;  Poisons  and  their 
Antidotes;  Weights  and  Measures;  Thermometric  Scales;  New 
Official  and  Unofficial  Drugs,  etc.  One  volume  of  over  800  pages. 
Prices,  with  Denison's  Patent  Ready-Reference  Index;  Cloth,  ^5.00 
net;  Sheep  or  Half  Morocco,  ^6.00  net;  Half  Russia,  $6.50  net. 
Without  Patent  Index:  Cloth,  $4.00  net;  Sheep  or  Half  Morocco, 
;^5.oo  net. 

'*  I  am  much  pleased  with  Keating's  Dictionary,  and  shall  take  pleasure  in  recommend- 
ing  it  to  my  classes." — Henry  M.  Lyman,  M.D.,  Professor  of  the  Principles  and  Practict 
of  Medicine,  Rush  Medical  College,  Chicago,  III. 

"  I  am  convinced  that  it  will  be  a  very  valuable  adjunct  to  my  study-table,  cpnvenient 
in  size  and  sufficiently  full  for  ordinary  use." — C.  A,  Lindsley,  M.D.,  Professor  of  the 
Theory  and  Practice  of  Medicine,  Medical  Dept.   Yale  University. 

KEATINQ»S   LIFE   INSURANCE. 

How  to  Examine  for  Life  Insurance.  By  John  M.  Keating, 
M.  D.,  Fellow  of  the  College  of  Physicians  of  Philadelphia;  Vice- 
President  of  the  American  Paediatric  Society;  Ex- President  of  thje 
Association  of  Life  Insurance  Medical  Directors.  Royal  octavo,  211 
pages ;  with  two  large  half-tone  illustrations,  and  a  plate  prepared  by 
Dr.  McClellan  from  special  dissections ;  also,  numerous  other  illustra- 
tions.    Cloth,  $2.00  net. 

"  This  is  by  far  the  most  useful  book  vv^hich  has  yet  appeared  on  insurance  examination, 
a  subject  of  growing  interest  and  importance.  Not  the  least  valuable  portion  of  the  volume 
is  Part  II.,  which  consists  of  instructions  issued  to  their  examining  physicians  by  twenty-four 
representative  companies  of  this  country.  If  for  these  alone,  the  book  should  be  at  the  right 
hand  of  every  physician  interested  in  this  special  branch  of  medical  science." — The  Medical 
News. 


Medical  Publications  of  W,  B.  Saunders,  15 

KEEN  ON  THE  SURGERY  OF  TYPHOID  FEVER. 

The   Surgical  Complications  and  Sequels  of  Typhoid    Fever. 

By  Wm.  W.  Keen,  M.D.,  LL.D.,  Professor  of  the  Principles  of  Sur- 
gery and  of  Clinical  Surgery,  Jefferson  Medical  College,  Philadelphia; 
Corresponding  Member  of  the  Societe  de  Chirurgie,  Paris ;  Honorary 
Member  of  the  Societe  Beige  de  Chirurgie,  etc.  Octavo  volume  of 
386  pages,  illustrated.     Cloth,  $3.00  net. 

"  This  is  probably  the  first  and  only  work  in  the  English  language  that  gives  the  reader 
a  clear  view  of  what  typhoid  fever  really  is,  and  what  it  does  and  can  do  to  the  human 
organism.  This  book  should  be  in  the  possession  of  every  medical  man  in  America." — 
American  Medico-Surgical  Bulletin. 

KEEN'S  OPERATION  BLANK.  Second  Edition,  Revised  Form. 
An  Operation  Blank,  with  Lists  of  Instruments,  etc.  Required 
in  Various  Operations.  Prepared  by  W.  W.  Keen,  M.D.,  LL.D., 
Professor  of  the  Principles  of  Surgery  in  Jefferson  Medical  College, 
Philadelphia.  Price  per  pad,  containing  blanks  for  fifty  operations, 
50  cents  net. 

KYLE  ON  THE  NOSE  AND  THROAT. 

Diseases  of  the  Nose  and  Throat.  By  D.  Braden  Kyle,  M.D., 
Clinical  Professor  of  Laryngology  and  Rhinology,  Jefferson  Medical 
College,  Philadelphia;  Consulting  Laryngologist,  Rhinologist,  and 
Otologist,  St.  Agnes'  Hospital.  Handsome  octavo  volume  of  about 
630  pages,  with  over  150  illustrations  and  6  lithographic  plates.  Price, 
Cloth,  114.00  net;   Half  Morocco,  $5.00  net. 

LAINE'S  TEMPERATURE  CHART. 

Temperature  Chart.  Prepared  by  D.  T.  Laine,  M.D.  Size  8  x  13^ 
inches.  A  conveniently  arranged  Chart  for  recording  Temperature, 
with  columns  for  daily  amounts  of  Urinary  and  Fecal  Excretions, 
Food,  Remarks,  etc.  On  the  back  of  each  chart  is  given  in  full  the 
method  of  Brand  in  the  treatment  of  Typhoid  Fever.  Price,  per  pad 
of  25  charts,  50  cents  net. 

"  To  the  busy  practitioner  this  chart  will  be  found  of  great  value  in  fever  cases,  and 
especially  for  cases  of  typhoid." — Indian  Lancet ^  Calcutta. 

lockwood's  practice  of  medicine. 

A  Manual  of  the  Practice  of  Medicine.  By  George  Roe  Lock- 
wood,  M.D.,  Professor  of  Practice  in  the  Woman's  Medical  College 
of  the  New  York  Infirmary,  etc..  935  pages,  with  75  illustrations  in 
the  text,  and  22  full-page  plates.     Cloth,  $2.50  net. 

"Gives  in  a  most  concise  manner  the  points  essential  to  treatment  usually  enumerated 
in  the  most  elaborate  works." — Massachusetts  Medical  Journal. 

LONG'S  SYLLABUS  OF  GYNECOLOGY. 

A  Syllabus  of  Gynecology,  arranged  in  Conformity  with  **  An 
American  Text=Book  of  Gynecology."  By  J.  W.  Long,  M.D., 
Professor  of  Diseases  of  Women  and  Children,  Medical  College  of 
Virginia,  etc.     Cloth,  interleaved,  $1.00  net. 

"  The  book  is  certainly  an  admirable  resumi  of  what  every  gynecological  student  and 
practitioner  should  know,  and  will  prove  of  value  not  only  to  those  who  have  the  '  American 
Text- Book  of  Gynecology,'  but  to  others  as  well." — Brooklyn  Medical  Journal. 


16  Medical  Publications  of  W.  B.  Saunders. 

MACDONALD'S  SURGICAL  DIAGNOSIS  AND  TREATMENT. 

Surgical  Diagnosis  and  Treatment.  By  J.  W.  Macdonald,  M.D. 
Edin.,  F.R.C.S.,  Edin.,  Professor  of  the  Practice  of  Surgery  and  of 
Clinical  Surgery  in  Hamline  University;  Visiting  Surgeon  to  St. 
Barnabas'  Hospital,  Minneapolis,  etc.  Handsome  octavo  volume  of 
800  pages,  profusely  illustrated.  Cloth,  $5.00  net;  Half  Morocco, 
;^6.oo  net. 

**  A  thorough  and  complete  work  on  surgical  diagnosis  and  treatment,  free  from  pad- 
ding, full  of  valuable  material,  and  in  accord  with  the  surgical  teaching  of  the  day." — T^e 
Medical  Nezvs,  New  York. 

•*  The  work  is  brimful  of  just  the  kind  of  'practical  information  that  is  useful  alike  to 
students  and  practitioners.  It  is  a  pleasure  to  commend  the  bock  because  of  its  intrinsic 
value  to  the  medical  practitioner." — Cincinnati  Lancet- Clinic . 

MALLORY  AND  WRIGHT'S  PATHOLOGICAL  TECHNIQUE. 

Pathological  Technique.  A  Practical  Manual  for  Laboratory  Work 
in  Pathology,  Bacteriology,  and  Morbid  Anatomy,  with  chapters  on 
Post-Mortem  Technique  and  the  Performance  of  Autopsies.  By  Frank 
B.  Mallory,  A.m.,  M.D.,  Assistant  Professor  of  Pathology,  Harvarn 
University  Medical  School,  Boston;  and  James  H.  Wright,  A.M., 
M.D.,  Instructor  in  Pathology,  Harvard  University  Medical  School, 
Boston.  Octavo  volume  of  396  pages,  handsomely  illustrated.  Cloth, 
;g2.5o  net. 

*'  I  have  been  looking  forward  to  the  publication  of  this  book,  and  I  am  glad  to  say  that 
I  find  it  to  be  a  most  useful  laboratory  and  post-mortem  guide,  full  of  practical,  information, 
and  well  up  to  date." — William  H.  Welch,  Professor  of  Pathology,  Johns  Hopkins  Uni- 
versity, Baltimore,  Md. 

MARTIN'S  MINOR  SURGERY,  BANDAGING,  AND  VENEREAL 
DISEASES.  Second  Edition,  Revised. 
Essentials  of  Minor  Surgery,  Bandaging,  and  Venereal 
Diseases.  By  Edward  Martin,  A.M.,  M.D.,  Clinical  Professor  of 
Genito-Urinary  Diseases,  University  of  Pennsylvania,  etc.  Crown 
octavo,  166  pages,  with  78  illustrations.  Cloth,  ^i.oo  j  interleaved  for 
notes,  ^1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"A  very  practical  and  systematic  study  of  the  subjects,  and  shows  the  author's  famil- 
iarity with  the  needs  of  students." — Therapeutic  Gazette. 

MARTIN'S  SURGERY.     Sixth  Edition,  Revised. 

Essentials  of  Surgery.  Containing  also  Venereal  Diseases,  Surgi- 
cal Landmarks,  Minor  and  Operative  Surgery,  and  a  complete  de- 
scription, with  illustrations,  of  the  Handkerchief  and  Roller  Bandages. 
By  Edward  Martin,  A.M.,  M.D.,  Clinical  Professor  of  Genito- 
Urinary  Diseases,  University  of  Pennsylvania,  etc.  Crown  octavo,  338 
pages,  illustrated.  With  an  Appendix  containing  full  directions  for  the 
preparation  of  the  materials  used  in  Antiseptic  Surgery,  etc.  Cloth, 
^i.oo;  interleaved  for  notes,  $1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

*'  Contains  all  necessary  essentials  of  modern  surgery  in  a  comparatively  small  space. 
Its  style  is  interesting,  and  its  illustrations  are  admirable." — Medical  and  Surgical  Reporter. 


Medical  Publications  of  W.  B.  Saunders.  17 

McFARLAND'S  PATHOGENIC  BACTERIA.  Second  Edition,  Re- 
vised and  Greatly  Enlarged. 
Text-Book  upon  the  Pathogenic  Bacteria.  By  Joseph  McFar- 
LAND,  M.  D. ,  Professor  of  Pathology  and  Bacteriology  in  the  Medico- 
Chirurgical  College  of  Philadelphia,  etc.  Octavo  volume  of  497  pages, 
finely  illustrated.     Cloth,  $2.50  net. 

**  Dr.  McFarland  has  treated  the  subject  in  a  systematic  manner,  and  has  succeeded  in 
presenting  in  a  concise  and  readable  form  the  essentials  of  bacteriology  up  to  date.  Alto- 
gether, the  book  is  a  satisfactory  one,  and  I  shall  take  pleasure  in  recommending  it  to  the 
students  of  Trinity  College."— H.  B.  Anderson,  M.D.,  Professor  of  Pathology  and  Bac- 
teriology^ Trinity  Medical  College,  Toronto. 

MEIGS  ON  FEEDING  IN  INFANCY. 

Feeding  in  Early  Infancy.  By  Arthur  V.  Meigs,  M.D.  Bound 
in  limp  cloth,  flush'edges,  25  cents  net. 

"  This  pamphlet  is  worth  many  times  over  its  price  to  the  physician.  The  author's 
experiments  and  conclusions  are  original,  and  have  been  the  means  of  doing  much  good." — 
Medical  Bulletin. 

MOORE'S  ORTHOPEDIC  SURGERY. 

A  Manual  of  Orthopedic  Surgery.  By  James  E.  Moore,  M.D., 
Professor  of  Orthopedics  and  Adjunct  Professor  of  Clinical  Surgery, 
University  of  Minnesota,  College  of  Medicine  and  Surgery.  Octavo 
volume  of  356  pages,  handsomely  illustrated.     Cloth,  ^2.50  net. 

"A  most  attractive  work.  The  illustrations  and  the  care  with  which  the  book  is  adapted 
to  the  wants  of  the  general  practitioner  and  the  student  are  worthy  of  great  praise." — Chicago 
Medical  Recorder. 

"A  very  demonstrative  work,  every  illustration  of  which  conveys  a  lesson.  The  work  is 
a  most  excellent  and  commendable  one,  which  we  can  certainly  endorse  with  pleasure." — 
St.  Louis  Medical  and  Surgical  Journal. 

MORRIS'S  MATERIA  MEDICA  AND  THERAPEUTICS.  Fifth 
Edition,  Revised. 
Essentials  of  Materia  Medica,  Therapeutics,  and  Prescription- 
Writing.  By  Henry  Morris,  M.D.,  late  Demonstrator  of  Thera- 
peutics, Jefferson  Medical  College,  Philadelphia;  Fellow  of  the  College 
of  Physicians,  Philadelphia,  etc.  Crown  octavo,  288  pages.  Cloth, 
;^i.oo;  interleaved  for  notes,  $1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"  This  work,  already  excellent  in  the  old  edition,  has  been  largely  improved  by  revi- 
sion." — American  Practitioner  and  News. 

MORRIS,  WOLFF,  AND  POWELL'S  PRACTICE  OF  MEDICINE. 
Third  Edition,  Revised. 
Essentials  of  the  Practice  of  Medicine.  By  Henry  Morris,  M.D., 
late  Demonstrator  of  Therapeutics,  Jefferson  Medical  College,  Phila- 
delphia ;  with  an  Appendix  on  the  Clinical  and  Microscopic  Examina- 
tion of  Urine,  by  Lawrence  Wolff,  M.D.,  Demonstrator  of  Chemistry, 
Jefferson  Medical  College,  Philadelphia.  Enlarged  by  some  300  essen- 
tial formulae  collected  and  arranged  by  William  M.  Powell,  M.D. 
Post-octavo,  488  pages.     Cloth,  ^2.00. 

[See  Saunders'  Question- Compends,  page  21.] 

"  The  teaching  is  sound,  the  presentation  graphic  ;  matter  full  as  can  be  desired,  and 
style  attractive." — American  Practitioner  and  News. 


18  Medical  Publications  of  W.  B,  Saunders. 

MORTEN'S  NURSE'S  DICTIONARY. 

Nurse's  Dictionary  of  Medical  Terms  and  Nursing  Treat- 
ment. Containing  Definitions  of  the  Principal  Medical  and  Nursing 
Terms  and  Abbreviations ;  of  the  Instruments,  Drugs,  Diseases,  Acci- 
dents, Treatments,  Operations,  Foods,  Appliances,  etc.  encountered 
in  the  ward  or  in  the  sick-room.  By  Honnor  Morten,  author  of 
^'  How  to  Become  a  Nurse,"  etc.     i6mo,  140  pages.     Cloth,  ^i.oo. 

**  A  handy,  compact  little  volume,  containing  a  large  amount  of  general  information,  all 
of  which  is  arranged  in  dictionary  or  encyclopedic  form,  thus  facilitating  quick  reference. 
It  is  certainly  of  value  to  those  for  whose  use  it  is  published." — Chicago  Clinical  Review. 

NANCREDE'S  ANATOMY.  Sixth  Edition,  Thoroughly  Revised. 
Essentials  of  Anatomy,  including  the  Anatomy  of  the  Viscera. 
By  Charles  B.  Nancrede,  M.D.,  LL.D.,  Professor  of  Surgery  and 
of  Clinical  Surgery  in  the  University  of  Michigan,  Ann  Arbor.  Crown 
octavo,  420  pages;  151  illustrations.  Based  upon  Gray's  Anatomy. 
Cloth,  $1.00  net;  interleaved  for  notes,  $1.25  net. 

[See  Saunders'  Question- Compends,  page  21.] 

"  For  self-quizzing  and  keeping  fresh  in  mind  the  knowledge  of  anatomy  gained  at 
school,  it  would  not  be  easy  to  speak  of  it  in  terms  too  favorable." — American  Practitioner. 

NANCREDE'S  ANATOMY  AND  DISSECTION.     Fourth  Edition. 
Essentials  of  Anatomy  and   Manual  of   Practical   Dissection. 

By  Charles  B.  Nancrede,  M.D.,  LL.D.,  Professor  of  Surgery  and  of 
Clinical  Surgery,  University  of  Michigan,  Ann  Arbor.  Post-octavo  ; 
500  pages,  with  full-page  lithographic  plates  in  colors,  and  nearly  200 
illustrations.     Extra  Cloth  (or  Oilcloth  for  dissection-room),  ^2.00  net. 

"  It  may  in  many  respects  be  considered  an  epitome  of  Gray's  popular  work  on  general 
anatomy,  at  the  same  time  having  some  distinguishing  characteristics  of  its  own  to  commend 
it.  The  plates  are  of  more  than  ordinary  excellence,  and  are  of  especial  value  to  students 
in  their  work  in  the  dissecting  room." — Journal  of  the  American  Medical  Association. 

NORRIS'S  SYLLABUS  OF  OBSTETRICS.  Third  Edition,  Revised. 
Syllabus  of  Obstetrical  Lectures  in  the  Medical  Department 
of  the  University  of  Pennsylvania.  By  Richard  C.  Norris, 
A.M.,  M.D.,  Demonstrator  of  Obstetrics,  University  of  Pennsylvania. 
Crown  octavo,  222  pages.      Cloth,  interleaved  for  notes,  $2.00  net. 

"This  work  is  so  far  superior  to  others  on  the  same  subject  that  we  take  pleasure  in 
calling  attention  briefly  to  its  excellent  features.  It  covers  the  subject  thoroughly,  and  will 
prove  invaluable  both  to  the  student  and  the  practitioner." — Medical  Record,  New  York. 

PENROSE'S  DISEASES  OF  WOMEN.     Second  Edition,  Revised. 
A  Text=Book  of  Diseases  of  Women.     By  Charles  B.  Penrose, 
M.D.,  Ph.D.,  Professor  of  Gynecology  in  the  University  of  Pennsyl- 
vania;   Surgeon    to   the    Gynecean    Hospital,    Philadelphia.     Octavo 
volume  of  529  pages,  handsomely  illustrated.     Cloth,  ^3.50  net. 

**  I  shall  value  very  highly  the  copy  of  Penrose's  *  Diseases  of  Women*  received. 
I  have  already  recommended  it  to  my  class  as  THE  BEST  book." — Howard  A.  KELLY, 
Professor  of  Gynecology  and  Obstetrics,  Johns  Hopkins  University,  Baltimore,  Md. 

*<  The  book  is  to  be  commended  without  reserve,  not  only  to  the  student  but  to  the 
general  practitioner  who  wishes  to  have  the  latest  and  best  modes  of  treatment  explained 
with  absolute  clearness." — Therapeutic  Gazette. 


Medical  Publications  of  W.  B,  Saunders.  19 

POWELL*S  DISEASES  OF  CHILDREN.     Second  Edition. 

Essentials  of  Diseases  of  Children.  By  William  M.  Powell, 
M.D.,  Attending  Physician  to  the  Mercer  House  for  Invalid  Women 
at  Atlantic  City,  N.  J. ;  late  Physician  to  the  Clinic  for  the  Diseases  of 
Children  in  the  Hospital  of  the  University  of  Pennsylvania.  Crown 
octavo,  222  pages.     Cloth,  ;^i.oo;  interleaved  for  notes,  ^1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

"Contains  the  gist  of  all  the  best  works  in  the  department  to  which  it  relates."— 
American  Practitioner  and  News. 

PRINGLE'S  SKIN  DISEASES  AND  SYPHILITIC  AFFECTIONS. 
Pictorial  Atlas  of  Skin  Diseases  and  Syphilitic  Affections 
(American  Edition).  Translation  from  the  French.  Edited  by 
J.  J.  Pringle,  M.B.,  F.R.C.P.,  Assistant  Physician  to  the  Middlesex 
Hospital,  London.  Photo-lithochromes  from  the  famous  models  in 
the  Museum  of  the  Saint-Louis  Hospital,  Paris,  with  explanatory  wood- 
cuts and  text.  In  12  Parts.  Price  per  Part,  $3.00.  Complete  in 
one  volume.  Half  Morocco  binding,  ^40.00  net. 

**  I  strongly  recommend  this  Atlas.  The  plates  are  exceedingly  well  executed,  and 
will  be  of  great  value  to  all  studying  dermatology." — Stephen  Mackenzie,  M.D. 

"The  introduction  of  explanatory  wood-cuts  in  the  text  is  a  novel  and  most  important 
feature  which  greatly  furthers  the  easier  understanding  of  the  excellent  plates,  than  which 
nothing,  we  venture  to  say,  has  been  seen  better  in  point  of  correctness,  beauty,  and  general 
merit." — New  York  Medical  Journal. 

PRYOR— PELVIC  INFLAMMATIONS. 

The  Treatment  of   Pelvic  Inflammations  through  the  Vagina. 

By  W.  R.  Pryor,  M.D.,  Professor  of  Gynecology  in  New  York  Poly- 
clinic.    i2mo,  248  pages,  handsomely  illustrated.     Cloth,  ^2.00  net. 

"  This  subject,  which  has  recently  been  so  thoroughly  canvassed  in  high  gynecological 
circles,  is  made  available  in  this  volume  to  the  general  practitioner  and  student.  Nothing  is 
too  minute  for  mention  and  nothing  is  taken  for  granted ;  consequently  the  book  is  of  the  utmost 
value.    The  illustrations  and  the  technique  are  beyond  criticism." — Chicago  Medical  Recorder^ 

PYE'S  BANDAGING. 

Elementary  Bandaging  and   Surgical  Dressing.      With  Direc- 
tions concerning  the  Immediate  Treatment  of  Cases  of  Emergency. 
For  the  use  of  Dressers  and  Nurses.     By  Walter  Pye,  F.R.C.S.,  late 
Surgeon  to  St.  Mary's  Hospital,  London.     Small  i2mo,  with  over  80 
illustrations.     Cloth,  flexible  covers,  75  cents  net. 
"The  directions  are  clear  and  the  illustrations  are  good." — London  Lancet. 
**  The  author  writes  well,  the  diagrams  are  clear,  and  the  book  itself  is  small  and  port- 
able, although  the  paper  and  type  are  good." — British  Medical  Journal. 

RAYMOND'S  PHYSIOLOGY. 

A  Manual  of  Physiology.  By  Joseph  H.  Raymond,  A.M.,  M.D., 
Professor  of  Physiology  and  Hygiene  and  Lecturer  on  Gynecology  in 
the  Long  Island  College  Hospital;  Director  of  Physiology  in  the 
Hoagland  Laboratory,  etc.  382  pages,  with  102  illustrations  in  the 
text,  and  4  full-page  colored  plates.     Cloth,  $1.25  net. 

"  Extremely  well  gotten  up,  and  the  illustrations  have  been  sel-ected  with  care.  The 
text  is  fully  abreast  with  modern  physiology.  "—^r//w>^  Medical  Journal, 


Arranged  in  Question  and 
Answer  Form> 

XJlJiO  1  VUi^  ntHE  MOST  COMPLETE  AND  BEST 

r^rMi;nD"civmc       illustrated  series  of 

^^'^•IVlJrlliNlJo  COMPENDS  EVER  ISSUED. 

Now  the  Standard  Authorities  in  Medical  Literature  . .  ♦  • 

with  Students  and  Practitioners  in  every  City  of  tlie  United  States  and  Canada* 


Saunders' 

Q 


^    OVER  175,000  COPIES  SOLD* 
THE  REASON  WHY* 

They  are  the  advance  guard  of  "Student's  Helps" — that  DO  help.  They  are  the 
leaders  in  their  special  line,  well  and  authoritatively  written  by  able  men,  who,  as  teachers  in 
the  large  colleges,  know  exactly  what  is  wanted  by  a  student  preparing  for  his  examinations. 
The  judgment  exercised  in  the  selection  of  authors  is  fully  demonstrated  by  their  professional 
standing.  Chosen  from  the  ranks  of  Demonstrators,  Quiz-masters,  and  Assistants,  most  of 
them  have  become  Professors  and  Lecturers  in  their  respective  colleges. 

Each  book  is  of  convenient  size  (5x7  inches) ,  containing  on  an  average  250  pages, 
profusely  illustrated,  and  elegantly  printed  in  clear,  readable  type,  on  fine  paper. 

The  entire  series,  numbering  twenty-three  volumes,  has  been  kept  thoroughly  revised 
and  enlarged  when  necessary,  many  of  the  books  being  in  their  fifth  and  sixth  editions. 

TO  SUM  UP* 

Although  there  are  numerous  other  Quizzes,  Manuals,  Aids,  etc.  in  the  market,  none  of 
them  approach  the  "  Blue  Series  of  Question  Compends;"  and  the  claim  is  made  for  the 
following  points  of  excellence  : 

1.  Professional  distinction  and  reputation  of  authors. 

2.  Conciseness,  clearness,  and  soundness  of  treatment. 

3.  Quality  of  illustrations,  paper,  printing,  and  binding. 

Any  cf  these  Compends  will  be  mailed  on  receipt  of  price  (see  next  page  for  List)* 


Saunders^  Question-Compend  Series* 

Price,  Qoth,  $J.OO  per  copy,  except  when  otherwise  noted. 

•'Where  the  work  of  preparing  students'  manuals  is  to  end  we  cannot  say,  but  the 
Saunders  Series,  in  our  opinion,  bears  off  the  palm  at  present."— A^(fzc/  York  Medical  Record. 


1.  ESSENTIALS  OF  PHYSIOLOGY.     By  H.  A.  Hare,  M.D.    Fourth  edition, 

revised  and  enlarged.      (^I.oo  net.) 

2.  ESSENTIALS  OF   SURGERY.     By  Edward  Martin,  M.D.      Sixth  edition, 

revised,  with  an  Appendix  on  Antiseptic  Surgery. 

3.  ESSENTIALS  OF   ANATOMY.      By  Chari.es  B.   Nancrede,  M.D.      Sixth 

edition,  thoroughly  revised  and  enlarged.     ($i.oo  net.) 

4.  ESSENTIALS  OF  MEDICAL  CHEMISTRY,  ORGANIC  AND  INORGANIC. 

By  Lawrence  Wolff,  M.D.     Fifth  edition,  revised.     ($i.oo  net.) 

5.  ESSENTIALS  OF  OBSTETRICS.     By  W.  Easterly  Ashton,  M.D.     Fourth 

edition,  revised  and  enlarged. 

6.  ESSENTIALS  OF  PATHOLOGY  AND  MORBID  ANATOMY.     By  C.  E. 

Armand  Semple,  M.D. 

7.  ESSENTIALS  OF  MATERIA  MEDICA,  THERAPEUTICS,  AND  PRE- 

SCRIPTION-WRITING.   By  Henry  Morris,  M.D.       Fifth  edition,  revised. 

8,9.  ESSENTIALS  OF  PRACTICE  OF  MEDICINE.  By  Henry  Morris, 
M.D.  An  Appendix  on  Urine  Examination.  By  Lawrence  Wolff,  M.D. 
Third  edition,  enlarged  by  some  300  Essential  Formulae,  selected  from  eminent 
authorities,  by  Wm.  M.  Powell,  M.D.     (Double  number,  ^2.00.) 

10.  ESSENTIALS  OF  GYNAECOLOGY.      By  Edwin  B.  Cragin,  M.D.      Fourth 

edition,  revised. 

11.  ESSENTIALS  OF  DISEASES  OF  THE  SKIN.     By  Henry  W.  Stelwagon, 

M.D.     Fourth  edition,  revised  and  enlarged,     (^i.oo  net.) 

12.  ESSENTIALS  OF  MINOR  SURGERY,  BANDAGING,  AND  VENEREAL 

DISEASES.     By  Edward  Martin,  M.D.     Second  ed.,  revised  and  enlarged. 

13.  ESSENTIALS  OF  LEGAL  MEDICINE,  TOXICOLOGY,  AND  HYGIENE. 

By  C.  E.  Armand  Semple,  M.D. 

14.  ESSENTIALS  OF   DISEASES  OF  THE   EYE,  NOSE,  AND  THROAT. 

By  Edward  Jackson,  M.D.,  and  E.  B.  Gleason,  M.D.     Second  ed.,  revised. 

15.  ESSENTIALS  OF  DISEASES  OF  CHILDREN.     By  William  M.  Powell, 

M.  D.     Second  edition. 

16.  ESSENTIALS  OF   EXAMINATION   OF   URINE.     By  Lawrence  Wolff, 

M.D.     Colored  **Vogel  Scale."     (75  cents.) 

17.  ESSENTIALS  OF  DIAGNOSIS.     By  s'.  Solis  Cohen,  M.D.,  and  A.  A.  Eshner, 

M.D.      (^1.50  net.) 

18.  ESSENTIALS  OF  PRACTICE  OF   PHARMACY.     By  Lucius  E.    Sayre. 

Second  edition,  revised  and  enlarged. 

20.  ESSENTIALS  OF  BACTERIOLOGY.     By  M.  V.  Ball,  M.D.     Third  edition, 

revised. 

21.  ESSENTIALS  OF  NERVOUS  DISEASES  AND  INSANITY.     By  John  C. 

Shaw,  M.D.     Third  edition,  revised. 

22.  ESSENTIALS  OF  MEDICAL  PHYSICS.      By  Fred  J.    Brockway,   M.D. 

Second  edition,  revised.      {^I.oo  net.) 

23.  ESSENTIALS  OF  MEDICAL  ELECTRICITY.    By  David  D.  Stewart,  M.D., 

and  Edward  S.  Lawrance,  M.D. 

24.  ESSENTIALS  OF  DISEASES  OF  THE   EAR.      By  E.  B.  Gleason,  M.D. 

Second  edition,  revised  and  greatly  enlarged. 


Pamphlet  containing  specimen  pagest  etc  sent  free  upon  application* 


Saunders' 
New  Series 


for  Students 


and 
of    Manuals  P'-actmoners. 


'TTtlAT  there  exists  a  need  for  thoroughly  reliable  hand-books  on  the  leading  branches 
of  Medicine  and  Surgery  is  a  fact  amply  demonstrated  by  the  favor  with  which 
the  SAUNDERS  NE^  SERIES  OF  MANUALS  have  been  received  by  medical 
students  and  practitioners  and  by  the  Medical  Press*  These  manuals  are  not  merely 
condensations  from  present  literature,  but  are  ably  written  by  w^ell-known  authors 
and  practitioners,  most  of  them  being  teachers  in  representative  American  colleges* 
Each  volume  is  concisely  and  authoritatively  written  and  exhaustive  in  detail,  without 
being  encumbered  with  the  introduction  of  **  cases,*'  w^hich  so  largely  expand  the 
ordinary  text-book.  These  manuals  will  therefore  form  an  admirable  collection  of 
advanced  lectures,  useful  alike  to  the  medical  student  and  the  practitioner:  to  the 
latter,  too  busy  to  search  through  page  after  page  of  elaborate  treatises  for  what  he 
grants  to  know,  they  w^ill  prove  of  inestimable  value ;  to  the  former  they  will  afford 
safe  guides  to  the  essential  points  of  study* 

The  SAUNDERS  NEW  SERIES  OF  MANUALS  are  conceded  to  be  superior 
to  any  similar  books  now  on  the  market.  No  other  manuals  afford  so  much  infor- 
mation in  such  a  concise  and  available  form.  A  liberal  expenditure  has  enabled  the 
publisher  to  render  the  mechanical  portion  of  the  work  worthy  of  the  high  literary 
standard  attained  by  these  books* 

Any  of  these  Manuals  will  be  mailed  on  receipt  of  price  (see  next  page  for  List)* 


Saunders^  New  Series  of  Manuals^ 


VOLUMES   PUBLISHED. 

PHYSIOLOGY.  By  Joseph  Howard  Raymond,  A.M.,  M.D.,  Professor  of  Physiology 
and  Hygiene  and  Lecturer  on  Gynecology  in  the  Long  Island  College  Hospital; 
Director  of  Physiology  in  the  Hoagland  Laboratory,  etc.     Illustrated.    Cloth,  ^1.25  net. 

SURGERY,  General  and  Operative.  By  John  Chalmers  DaCosta,  M.D.,  Clini- 
cal Professor  of  Surgery,  Jefferson  Medical  College,  Philadelphia;  Surgeon  to  the 
Philadelphia  Hospital,  etc.  Second  edition,  thoroughly  revised  and  greatly  enlarged. 
Octavo,  911  pages,  profusely  illustrated.     Cloth,  $4.00  net ;  Half  Morocco,  $5.00  net. 

DOSE-BOOK    AND    MANUAL    OF    PRESCRIPTION=WRITING.      By  E.    Q. 

Thornton,  M.D.,  Demonstrator  of  Therapeutics,  Jefferson*  Medical  College,  Phila- 
delphia.    Illustrated.     Cloth,  ^1.25  net. 

SURGICAL  ASEPSIS.  By  Carl  Beck,  M.D.,  Surgeon  to  St.  Mark's  Hospital  and 
to  the  New  York  German  PohlUinik,  etc.     Illustrated.     Cloth,  ^1.25  net. 

MEDICAL  JURISPRUDENCE.  By  Henry  C.  Chapman,  M.D.  Professor  of  Insti- 
tutes of  Medicine  and  Medical  Jurisprudence  in  the  Jefferson  Medical  College  of  Phila- 
delphia.    Illustrated.     Cloth,  ^1.50  net. 

SYPHILIS  AND  THE  VENEREAL  DISEASES.  By  James  Nevins  Hyde,  M.D., 
Professor  of  Skin  and  Venereal  Diseases,  and  Frank  H.  Montgomery,  M.D., 
Lecturer  on  Dermatology  and  Genito-Urinary  Diseases  in  Rush  Medical  College, 
Chicago.     Profusely  illustrated.     Cloth,  ^2.50  net. 

PRACTICE  OF  MEDICINE.  By  George  Roe  Lockwood,  M.D.,  Professor  of 
Practice  in  the  Woman's  Medical  College  of  the  New  York  Infirmary;  Instructor  in 
Physical  Diagnosis  in  the  Medical  Department  of  Columbia  College,  etc.  Illustrated. 
Cloth,  ^2.50  net. 

MANUAL  OF  ANATOMY.  By  Irving  S.  Haynes,  M.D.,  Adjunct  Professor  of 
Anatomy  and  Demonstrator  of  Anatomy,  Medical  Department  of  the  New  York 
University,  etc.     Beautifully  illustrated.     Cloth,  ^2.50  net. 

MANUAL  OF  OBSTETRICS.  By  W.  A.  Newman  Dorland,  M.D.,  Assistant 
Demonstrator  of  Obstetrics,  University  of  Pennsylvania ;  Chief  of  Gynecological  Dis- 
pensary, Pennsylvania  Hospital,  etc.     Profusely  illustrated.     Cloth,  ^2.50  net. 

DISEASES  OF  WOMEN.  By  J.  Bland  Sutton,  F.  R.  C.  S.,  Assistant  Surgeon  to 
Middlesex  Hospital  and  Surgeon  to  Chelsea  Hospital,  London;  and  Arthur  E. 
Giles,  M.  D.,  B.  Sc.  Lond.,  P\R.C.S.  Edin.,  Assistant  Surgeon  to  Chelsea  Hospital, 
London.     Handsomely  illustrated.     Cloth,  1^2.50  net. 


VOLUMES  IN  PREPARATION. 

NERVOUS  DISEASES.  By  Charles  W.  Burr,  M.D.,  Clinical  Professor  of  Nervous 
Diseases,  Medico-Chirurgical  College,  Philadelphia ;  Pathologist  to  the  Orthopaedic 
Hospital  and  Infirmary  for  Nervous  Diseases ;  Visiting  Physician  to  the  St.  Joseph 
Hospital,  etc. 

*#*  There  will  be  published  in  the  same  series,  at  short  intervals,  carefully-prepared  works 
on  various  subjects  by  prominent  specialists. 


Pamphlet  containiiig  specimen  pages,  etc*  sent  &ee  upon  application* 


24  Medical  Publications  of  W,  B,  Saunders. 

SAUNDBY'S  RENAL  AND  URINARY  DISEASES. 

Lectures  on  Renal  and  Urinary  Diseases.  By  Robert  Saundby, 
M.D.  Edin.,  Fellow  of  the  Royal  College  of  Physicians,  London,  and 
of  the  Royal  Medico-Chirurgical  Society ;  Physician  to  the  General 
Hospital ;  Consulting  Physician  to  the  Eye  Hospital  and  to  the  Hos- 
pital for  Diseases  of  Women;  Professor  of  Medicine  in  Mason  College, 
Birmingham,  etc.  Octavo  volume  of  434  pages,  with  numerous  illus- 
trations and  4  colored  plates.     Cloth,  $2.50  net. 

"  The  volume  makes  a  favorable  impression  at  once.  The  style  is  clear  and  succinct. 
We  cannot  find  any  part  of  the  subject  in  which  the  views  expressed  are  not  carefully  thought 
out  and  fortified  by  evidence  drawn  from  the  most  recent  sources.  The  book  may  be  cordially 
recommended.' ' — British  Medical  Journal. 

SAUNDERS'  MEDICAL  HAND=ATLASES. 

This  series  of  books  consists  of  authorized  translations  into  English  of 
the  world-famous  Lehmann  Medicinische  Handatlanten.  Each 
volume  contains  from  50  to  100  colored  lithographic  plates,  besides 
numerous  illustrations  in  the  text.  There  is  a  full  description  of  each 
plate,  and  each  book  contains  a  condensed  but  adequate  outline  of  the 
subject  to  which  it  is  devoted.  For  full  description  of  this  series,  with 
list  of  volumes  and  prices,  see  page  2. 

"  Lehmann  Medicinische  Handatlanten  belong  to  that  class  of  books  that  are  too  good 
to  be  appropriated  by  any  one  nation." — Journal  of  Eye,  Ear,  and  Throat  Diseases. 

"  The  appearance  of  these  works  marks  a  new  era  in  illustrated  English  medical 
works." — The  Canadian  Practitioner. 

SAUNDERS'   POCKET  MEDICAL   FORMULARY.      Fifth   Edition, 
Revised. 

By  William  M.  Powell,  M.D.,  Attending  Physician  to  the  Mercer 
House  for  Invalid  Women  at  Atlantic  City,  N.  J.  Containing  1800 
formulae  selected  from  the  best-known  authorities.  With  an  Appen- 
dix containing  Posological  Table,  Formulae  &nd  Doses  for  Hypo- 
dermic Medication,  Poisons  and  their  Antidotes,  Diameters  of  the 
Female  Pelvis  and  Foetal  Head,  Obstetrical  Table,  Diet  List  for  Various 
Diseases,  Materials  and  Drugs  used  in  Antiseptic  Surgery,  Treatment 
of  Asphyxia  from  Drowning,  Surgical  Remembrancer,  Tables  of 
Incompatibles,  Eruptive  Fevers,  Weights  and  Measures,  etc.  Hand- 
somely bound  in  flexible  morocco,  with  side  index,  wallet,  and  flap. 
;^i.75net. 

"  This  little  book,  that  can  be  conveniently  carried  in  the  pocket,  contains  an  immense 
amount  of  material.  It  is  very  useful,  and,  as  the  name  of  the  author  of  each  prescription 
is  given,  is  unusually  reliable." — Medical  Record,  New  York. 

SAYRE'S  PHARMACY.     Second  Edition,  Revised. 

Essentials  of  the  Practice  of  Pharmacy.  By  Lucius  E.  Sayre, 
M.D.,  Professor  of  Pharmacy  and  Materia  Medica  in  the  University  of 
Kansas.  Crown  octavo,  200  pages.  Cloth,  $1.00;  interleaved  for 
notes,  $1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

'*The  topics  are  treated  in  a  simple,  practical  manner,  and  the  work  forms  a  very  useful 
student's  manual." — Boston  Medical  and  Surgical  Journal. 


Medical  Publications  of  W.  B,  Saunders.  25 

SEMPLE'S  LEGAL  MEDICINE,  TOXICOLOGY,  AND  HYGIENE. 
Essentials  of   Legal   Medicine,  Toxicology,  and  Hygiene.     By 

C.  E.  Armand  Semple,  B.  A.,  M.  B.  Cantab.,  M.  R.  C.  P.  Lond., 
Physician  to  the  Northeastern  Hospital  for  Children,  Hackney,  etc. 
Crown  octavo,  212  pages;  130  illustrations.  Cloth,  ;^i. 00;  interleaved 
for  notes,  ^1.25. 

[See  Saunders'  Question- Compends,  page  21.] 

•'  No  general  practitioner  or  student  can  afford  to  be  without  this  valuable  work.  The 
subjects  are  dealt  with  by  a  masterly  hand." — London  Hospital  Gazette. 

SEMPLE'S  PATHOLOGY  AND  MORBID  ANATOMY. 

Essentials    of    Pathology    and    Morbid    Anatomy.      By  C.   E. 

Armand  Semple,  B.A.,  M.B.  Cantab.,  M.R.C.P.  Lond.,  Physician  to 
the  Northeastern  Hospital  for  Children,  Hackney,  etc.     Crown  octavo, 
174  pages;  illustrated.     Cloth,  $1.00;  interleaved  for  notes,  $1.25. 
[See  Saunders'  Question- Compends,  page  21.] 

"  Should  take  its  place  among  the  standard  volumes  on  the  bookshelf  of  both  student 
and  practitioner." — London  Hospital  Gazette. 

SENN'S  GENITO-URINARY  TUBERCULOSIS. 

Tuberculosis  of  the  Genito-Urinary  Organs,  Male  and  Female. 

By  Nicholas  Senn,  M.D.,  Ph.D.,  LL.D.,  Professor  of  the  Practice  of 
Surgery  and  of  Clinical  Surgery,  Rush  Medical  College,  Chicago. 
Handsome  octavo  volume  of  320  pages,  illustrated.     Cloth,  ^3.00  net. 

*'  An  important  book  upon  an  important  subject,  and  written  by  a  man  of  mature  judg- 
ment and  wide  experience.  The  author  has  given  us  an  instructive  book  upon  one  of  the 
most  important  subjects  of  the  day." — Clinical  Reporter. 

"  A  work  which  adds  another  to  the  many  obHgations  the  profession  owes  the  talented 
author." — Chicago  Medical  Recorder. 

SENN'S  SYLLABUS  OF  SURGERY. 

A  Syllabus  of  Lectures  on  the  Practice  of  Surgery,  arranged 
in  conformity  with  **  An  American  Text-Book  of  Surgery."    By 

Nicholas  Senn,  M.D.,  Ph.D.,  Professor  of  the  Practice  of  Surgery  and 
of  Clinical  Surgery  in  Rush  Medical  College,  Chicago.     Cloth,  ^2.00. 

"  This  syllabus  will  be  found  of  service  by  the  teacher  as  well  as  the  student,  the  work 
being  superbly  done.  There  is  no  praise  too  high  for  it.  No  surgeon  should  be  without 
it." — New  York  Medical  Times. 

SExNN'S  TUMORS. 

Pathology  and  Surgical  Treatment  of  Tumors.     By  N.  Senn, 

M.D.,  Ph.D.,  LL.D.,  Professor  of  Surgery  and  of  Clinical  Surgery, 
Rush  Medical  College ;  Professor  of  Surgery,  Chicago  Polyclinic ; 
Attending  Surgeon  to  Presbyterian  Hospital ;  Surgeon-in-Chief,  St. 
Joseph's  Hospital,  Chicago.  Octavo  volume  of  710  pages,  with  515 
engravings,  including  full-page  colored  plates.  New  and  Revised  Edi- 
tion in  Preparation. 

**  The  most  exhaustive  of  any  recent  book  in  Engnsh  on  this  subject.  It  is  well  illus- 
trated, and  will  doubtless  remain  as  the  principal  monograph  on  the  subject  in  our  language 
for  some  years.  The  book  is  handsomely  illustrated  and  printed,  and  the  author  has  giyen  a 
notable  and  lasting  contribution  to  surgery." — Journal  of  the  American  Medical  Association. 


Medical  Publications  of  W.  B,  Saunders, 


SHAW'S  NERVOUS  DISEASES  AND  INSANITY.  Third  Edition, 
Revised. 
Essentials  of  Nervous  Diseases  and  Insanity.  By  John  C. 
Shaw,  M.D.,  Clinical  Professor  of  Diseases  of  the  Mind  and  Nervous 
System,  Long  Island  College  Hospital  Medical  School ;  Consulting 
Neurologist  to  St.  Catherine's  Hospital  and  to  the  Long  Island  College 
Hospital.  Crown  octavo,  i86  pages;  48  original  illustrations.  Cloth, 
^i.oo  ;  interleaved  for  notes,  $1.25. 

[See  Saunders'  Question- Compendsy  page  21.] 

"Clearly  and  intelligently  written." — Boston  Medical  and  Surgical  Journal. 

"  There  is  a  mass  of  valuable  material  crowded  into  this  small  compass." — American 
Medico- Surgical  Bulletin. 

STARR'S  DIETS  FOR  INFANTS  AND  CHILDREN. 

Diets  for  Infants  and  Children  in  Health  and  in  Disease.     By 

Louis  Starr,  M.D.,  Editor  of  ''An  American  Text-Book  of  the 
Diseases  of  Children."  230  blanks  (pocket-book  size),  perforated 
and  neatly  bound  in  flexible  morocco.     $1.25  net. 

The  first  series  of  blanks  are  prepared  for  the  first  seven  months  of  infant  life  ;  each 
blank  indicates  the  ingredients,  but  not  the  quantities,  of  the  food,  the  latter  directions  being 
left  for  the  physician.  After  the  seventh  month,  modifications  being  less  necessary,  the  diet 
lists  are  printed  in  full.     Formulae  for  the  preparation  of  diluents  and  foods  are  appended. 

STELW AGON'S  DISEASES  OF  THE  SKIN.  Fourth  Ed.,  Revised. 
Essentials  of  Diseases  of  the  Skin.  By  Henry  W.  Stelwagon, 
M.D.,  Clinical  Professor  of  Dermatology  in  the  Jefferson  Medical 
College,  Philadelphia;  Dermatologist  to  the  Philadelphia  Hospital; 
Physician  to  the  Skin  Department  of  the  Howard  Hospital,  etc. 
Crown  octavo,  276  pages;  88  illustrations.  Cloth,  $1.00  net;  inter- 
leaved for  notes,  $1.25  net. 

[See  Saunders''  Question- Co7npends,  page  21.] 
**  The  best  student's  manual  on  skin  diseases  we  have  yet  seen." — Times  and  Register^ 

STENGEL'S  PATHOLOGY.      Second  Edition. 

A  Text-Book  of  Pathology.  By  Alfred  Stengel,  M.D.,  Professor 
of  Clinical  Medicine  in  the  University  of  Pennsylvania ;  Physician  to 
the  Philadelphia  Hospital ;  Physician  to  the  Children's  Hospital,  etc. 
Handsome  octavo  volume  of  848  pages,  with  nearly  400  illustrations, 
many  of  them  in  colors.  Cloth,  ^4.00  net;  Half  Morocco,  ^5.00 
net. 

STEVENS'  MATERIA   MEDIC  A   AND   THERAPEUTICS.      Second 
Edition,  Revised. 
A  Manual  of   Materia   Medica   and  Therapeutics.      By  A.  A. 

Stevens,  A.M.,  M.D.,  Lecturer  on  Terminology  and   Instructor  in 
Physical  Diagnosis   in  the  University  of  Pennsylvania;    Professor  of 
Pathology  in  the  Woman's  Medical  College  of  Pennsylvania.     Post- 
octavo,  445  pages.     Flexible  leather,  ^2.25. 
"The  author  has  faithfully  presented  modern  therapeutics  in  a  comprehensive  work, 
and,  while  intended  particularly  for  the  use  of  students,  it  will  be  found  a  reliable  guide  and 
sufficiently  comprehensive  for  the  physician  in  practice." — University  Medical  Magazine, 


Medical  Publications  of  W.  B.  Saunders.  27 

5TEVENS'  PRACTICE  OF  MEDICINE.  Fifth  Edition,  Revised. 
A  Manual  of  the  Practice  of  Medicine.  By  A.  A.  Stevens,  A.  M., 
M.  D.,  Lecturer  on  Terminology  and  Instructor  in  Physical  Diagnosis 
in  the  University  of  Pennsylvania;  Professor  of  Pathology  in  the 
Woman's  Medical  College  of  Pennsylvania.  Specially  intended  for 
students  preparing-  for  graduation  and  hospital  examinations.  Post- 
octavo,  519  pages;   illustrated.     Flexible  leather,  ^2.00  net. 

"  The  frequency  with  which  new  editions  of  this  manual  are  demanded  bespeaks  its 
popularity.  It  is  an  excellent  condensation  of  the  essentials  of  medical  practice  for  the 
student,  and  maybe  found  also  an  excellent  reminder  for  the  busy  physician." — Buffalo 
Medical  Journal. 

STEWARTS  PHYSIOLOGY.      Third  Edition,  Revised. 

A  Manual  of  Physiology,  with  Practical  Exercises.  For 
Students  and  Practitioners.  By  G.  N.  Stewart,  M.A.,  M.D., 
D.Sc,  lately  Examiner  in  Physiology,  University  of  Aberdeen,  and 
of  the  New  Museums,  Cambridge  University ;  Professor  of  Physiology 
in  the  Western  Reserve  University,  Cleveland,  Ohio.  Octavo  volume 
of  848  pages;  300  illustrations  in  the  text,  and  5  colored  plates. 
Cloth,  $3.75  net. 

**  It  will  ma,ke  its  way  by  sheer  force  of  merit,  and  amply  deserves  to  do  so.  It  is  one 
of  the  very  best  English  tekt-books  on  the  subject." — London  Lancet. 

'  *  Of  the  many  text-books  of  physiology  published,  we  do  not  know  of  one  that  so 
nearly  comes  up  to  the  ideal  as  does  Prof.  Stewart's  volume." — British  Medical  Journal. 

STEWART  AND  LAWRANCE'S  MEDICAL  ELECTRICITY. 

Essentials  of  Medical  Electricity.  By  D.  D.  Stewart,  M.D., 
Demonstrator  of  Diseases  of  the  Nervous  System  and  Chief  of  the 
Neurological  Clinic  in  the  Jefferson  Medical  College;  and  E.  S. 
Lawrance,  M.D.,  Chief  of  the  Electrical  Clinic  and  Assistant  Demon- 
strator of  Diseases  of  the  Nervous  System  in  the  Jefferson  Medical 
College,  etc.  Crown  octavo,  158  pages;  65  illustrations.  Cloth, 
^i.oo;  interleaved  for  notes,  ^1.25. 

[See  Saunders'  Question- Compends^  page  21.] 

**  Throughout  the  whole  brief  space  at  their  command  the  authors  show  a  discriminating 
knowledge  of  their  subject." — Medical  News. 

STONEY'S  NURSING.     Second  Edition,  Revised. 

Practical  Points  in  Nursing.     For  Nurses  in  Private  Practice. 

By  Emily  A.  M.  Stoney,  Graduate  of  the  Training-School  for  Nurses, 
Lawrence,  Mass.;  late  Superintendent  of  the  Training-School  for 
Nurses,  Carney  Hospital,  South  Boston,  Mass.  456  pages,  illustrated 
with  73  engravings  in  the  text,  and  8  colored  and  half-tone  plates. 
Cloth,  1 1. 75  net. 

"  There  are  few  books  intended  for  non-professional  readers  which  can  be  so  cordially 
endorsed  by  a  medical  journal  as  can  this  one." — Therapeutic  Gazette. 

"  This  is  a  well-written,  eminently  practical  volume,  which  covers  the  entire  range  of 
private  nursing  as  distinguished  from  hospital  nursing,  and  instructs  the  nurse  how  best  to 
meet  the  various  emergencies  which  may  arise,  and  how  to  prepare  everything  ordinarily 
needed  in  the  illness  of  her  patient." — American  Journal  of  Obstetrics  and  Diseases  of 
Women  and  Childi'en. 

"  It  is  a  work  that  the  physician  can  place  in  the  hands  of  his  private  nurses  with  the 
assurance  of  benefit," — Ohio  Medical  Journal. 


28  Medical  Publications  of  W.  B.  Saunders. 


STONEY'S  MATERIA   MEDICA   FOR  NURSES. 

Materia  Medica  for  Nurses.  By  Emily  A.  M.  Stoney,  Graduate  of 
the  Training-School  for  Nurses,  Lawrence,  Mass.  ;  late  Superintendent 
of  the  Training-School  for  Nurses,  Carney  Hospital,  South  Boston,  Mass. 
Handsome  octavo  volume  of  306  pages.     Cloth,  ;^i.5o  net. 

The  present  book  differs  from  other  similar  works  in  several  features,  all  of  which  are 
intended  to  render  it  more  practical  and  generally  useful.  The  general  plan  of  the  contents 
follows  the  lines  laid  down  in  training-schools  for  nurses,  but  the  book  contains  much  use- 
ful matter  not  usually  included  in  works  of  this  character,  such  as  Poison-emergencies, 
Ready  Dose-list,  Weights  and  Measures,  etc.,  as  well  as  a  Glossary,  defining  all  the  terms 
used  in  Materia  Medica,  and  describing  all  the  latest  drugs  and  remedies,  which  have  been 
generally  neglected  by  other  books  of  the  kind. 

SUTTON  AND  GILES'  DISEASES  OF  WOMEN. 

Diseases  of  Women.  By  J.  Bland  Sutton,  F.R.C.S.,  Assistant 
Surgeon  to  Middlesex  Hospital,  and  Surgeon  to  Chelsea  Hospital, 
London;  and  Arthur  E.  Giles,  M.D.,  B.Sc.  Lond.,  F.R.C.S.  Edin., 
Assistant  Surgeon  to  Chelsea  Hospital,  London.  436  pages,  hand- 
somely illustrated.     Cloth,  ^2.50  net. 

"The  text  has  been  carefully  prepared.  Nothing  essential  has  been  omitted,  and  its 
teachings  are  those  recommended  by  the  leading  authorities  of  the  day." — Journal  of  the 
American  Medical  Association. 

THOMAS'S  DIET  LISTS  AND  SICK=ROOM  DIETARY. 

Diet  Lists  and  Sick=Room  Dietary.  By  Jerome  B.  Thomas, 
M.D.,  Visiting  Physician  to  the  Home  for  Friendless  Women  and 
Children  and  to  the  Newsboys'  Home  ;  Assistant  Visiting  Physician 
to  the  Kings  County  Hospital.     Cloth,  $1.50.     Send  for  sample  sheet. 

THORNTON'S  DOSE=BOOK  AND  PRESCRIPTION-WRITING. 

Dose=Book  and  Manual  of   Prescription=Writing.      By  E.    Q. 

Thornton,  M.D.,  Demonstrator  of  Therapeutics,  Jefferson  Medical 
College,  Philadelphia.      334  pages,  illustrated.      Cloth,  $1.25  net. 

"Full  of  practical  suggestions;  will  take  its  place  in  the  front  rank  of  works  of  this 
sort. " — Medical  Record^  New  York. 

VAN  VALZAH  AND  NISBET'S  DISEASES  OF  THE  STOMACH. 
Diseases  of  the  Stomach.  By  William  W.  Van  Valzah,  M.D., 
Professor  of  General  Medicine  and  Diseases  of  the  Digestive  System 
and  the  Blood,  New  York  Polyclinic;  and  J.  Douglas  Nisbet,  M.D., 
Adjunct  Professor  of  General  Medicine  and  Diseases  of  the  Digestive 
System  and  the  Blood,  New  York  Polyclinic.  Octavo  volume  of  674 
pages,  illustrated.     Cloth,  $3.50  net. 

"  Its  chief  claim  lies  in  its  clearness  and  general  adaptability  to  the  practical  needs  of 
the  general  practitioner  or  student.  In  these  relations  it  is  probably  the  best  of  the  recent 
special  works  on  diseases  of  the  stomach." — Chicago  Clinical  Review. 

VECKrS  SEXUAL  IMPOTENCE. 

The  Pathology  and  Treatment  of  Sexual  Impotence.  By  Victor 
G.  Vecki,  M.D.  From  the  second  German  edition,  revised  and  en- 
larged.    Demi-octavo,  about  300  pages.     Cloth,  ^2.00  net. 

The  subject  of  impotence  has  seldom  been  treated  in  this  country  in  the  truly  scientific 
spirit  that  it  deserves.  Dr.  Vecki's  work  has  long  been  favorably  known,  and  the  German 
txx)k  has  received  the  highest  consideration.  This  edition  is  more  than  a  mere  translation, 
for,  although  based  on  the  German  edition,  it  has  been  entirely  rewritten  in  English. 


Medical  Publications  of  W,  B.  Saunders,  29 

VIERORDT'S  MEDICAL  DIAGNOSIS.  Fourth  Edition,  Revised. 
Medical  Diagnosis.  By  Dr.  Oswald  Vierordt,  Professor  of  Medi- 
cine at  the  University  of  Heidelberg.  Translated,  with  additions, 
from  the  fifth  enlarged  German  edition,  with  the  author's  permission, 
by  Francis  H.  Stuart,  A.  M.,  M.  D.  Handsome  royal  octavo  volume 
of  603  pages;  194  fine  wood-cuts  in  text,  many  of  them  in  colors. 
Cloth,  $4.00  net;  Sheep  or  Half  Morocco,  $5.00  net. 

"  A  treasury  of  practical  information  which  will  be  found  of  daily  use  to  every  busy 
practitioner  who  will  consult  it." — C.  A.  Lindsley,  M.D.,  Professor  of  the  Theory  and 
Practice  of  Medicine^  Yale  University. 

"  Rarely  is  a  book  published  with  which  a  reviewer  can  find  so  little  fault  as  with  the 
volume  before  us.  Each  particular  item  in  the  consideration  of  an  organ  or  apparatus,  which 
is  necessary  to  determine  a  diagnosis  of  any  disease  of  that  organ,  is  mentioned ;  nothing 
seems  forgotten.  The  chapters  on  diseases  of  the  circulatory  and  digestive  apparatus  and 
nervous  system  are  especially  full  and  valuable.  The  reviewer  would  repeat  that  the  book  is 
one  of  the  best — probably  the  best — which  has  fallen  into  his  hands." — University  Medical 
Magazine. 

WARREN'S  SURGICAL  PATHOLOGY  AND  THERAPEUTICS. 

Surgical  Pathology  and  Therapeutics.  By  John  Collins  Warren, 
M.D.,  LL.D.,  Professor  of  Surgery,  Medical  Department  Harvard 
University;  Surgeon  to  the  Massachusetts  General  Hospital,  etc. 
Handsome  octavo  volume  of  832  pages;  136  relief  and  lithographic 
illustrations,  33  of  which  are  printed  in  colors,  and  all  of  which  were 
drawn  by  William  J.  Kaula  from  original  specimens.  Revised  and 
Enlarged  Edition  in  Preparation. 

"There  is  the  work-  of  Dr.  Warren,  which  I  think  is  the  most  creditable  book  on 
Surgical  Pathology,  and  the  most  beautiful  medical  illustration  of  the  bookmaker's  art,  that 
has  ever  been  issued  from  the  American  press." — Dr.  Roswell  Park,  in  the  Hai-vard 
Graduate  Magazine. 

"  The  handsomest  specimen  of  bookmaking  that  has  ever  been  issued  from  the  American 
medical  press." — Atnerican  Journal  of  the  Medical  Sciences. 

*'  A  most  striking  and  very  excellent  feature  of  this  book  is  its  illustrations.  Without 
exception,  from  the  point  of  accuracy  and  artistic  merit,  they  are  the  best  ever  seen  in  a  work 
of  this  kind.  Many  of  those  representing  microscopic  pictures  are  so  perfect  in  their  coloring 
and  detail  as  almost  to  give  the  beholder  the  impression  that  he  is  looking  down  the  barrel 
of  a  microscope  at  a  welbmounted  section." — Annals  of  Surgery. 

WOLFF  ON  EXAMINATION  OF  URINE. 

Essentials  of  Examination  of  Urine.  By  Lawrence  Wolff,  M.D., 
Demonstrator  of  Chemistry,  Jefferson  Medical  College,  Philadelphia, 
etc.  Colored  (Vogel)  urine  scale  and  numerous  illustrations.  Crown 
octavo.      Cloth,  75  cents. 

[See  Saunders''  Question- Compends,  page  21.] 
**  A  very  good  work  of  its  kind — very  well  suited  to  its  purpose." — Titties  and  Register. 

WOLFF'S  MEDICAL  CHEMISTRY.     Fifth  Edition,  Revised. 

Essentials    of    Medical    Chemistry,   Organic    and    Inorganic. 

Containing  also  Questions  on  Medical  Physics,  Chemical  Physiology, 
Analytical  Processes,  Urinalysis,  and  Toxicology.  By  Lawrence 
Wolff,  M.D.,  Demonstrator  of  Chemistry,  Jefferson  Medical  College, 
Philadelphia,  etc.  Crown  octavo,  222  pages.  Cloth,  $1.00  net;  inter- 
leaved for  notes,  $1.25  net. 

[See  Saunders'  Question- Compcnds,  page   21.] 

"The  scope  of  this  work  is  certainly  equal  to  that  of  the  best  course  of  lectures  on 
Medical  Chemistry." — Fharvtaceutical  Era. 


CLASSIFIED    LIST 


Medical  Publications 


W.  B.  SAUNDERS, 

925  Walnut  Street,  PhiladelpKia* 


ANATOMY,  EMBRYOLOGY, 
HISTOLOGY. 

Clarkson — A  Text-Book  of  Histology,  9 

Haynes — A  Manual  of  Anatomy,  ...  13 

Heisler — A  Text- Book  of  Embryology,  1 3 

Nancrede — Essentials  of  Anatomy,  .  .  i8 
Nancrede — Essentials  of  Anatomy  and 

Manual  of  Practical  Dissection,  ...  18 
Semple — Essentials   of  Pathology  and 

Morbid  Anatomy 25 

BACTERIOLOGY. 

Ball — Essentials  of  Bacteriology,  ...      6 
Crookshank — A  Text-Book  of  Bacteri- 
ology,   10 

Frothingham— Laboratory  Guide,  .  .  II 
Mallory   and    Wright  —  Pathological 

Technique, 16 

McFarland — Pathogenic  Bacteria,    .    .    17 

CHARTS,  DIET-LISTS,  ETC. 

Griffith — Infant's  Weight  Chart,    ...  12 

Hart — Diet  in  Sickness  and  in  Health,  .  13 

Keen — Operation  Blank, 15 

Laine — Temperature  Chart, 15 

Meigs^ — Feeding  in  Eariy  Infancy,     .    .  17 

Starr — Diets  for  Infants  and  Children,  .  26 
Thomas — Diet-Lists     and    Sick-Room 

Dietary, 28 

CHEMISTRY  AND  PHYSICS. 

Brockway — Essentials  of  Medical  Phys- 
ics,        7 

Wolff — Essentials  of  Medical  Chemistry,  29 

CHILDREN. 

An  American  Text-Book  of  Diseases 

of  Children,   . 3 

Griffith — Care  of  the  Baby, 12 

Griffith — Infant's  Weight  Chart,  ...  12 

Meigs — Feeding  in  Early  Infancy,    .    .  17 

Powell — Essentials  of  Dis.  of  Children,  19 

Starr — Diets  for  Infants  and  Children,  .  26 

DIAGNOSIS. 

Cohen  and  Eshner— Essentials  of  Di- 
agnosis,     .      9 

Corwin — Physical  Diagnosis,      ....      9 

Macdonald — Surgical  Diagnosis  and 
Treatment, l6 

Vierordt — Medical  Diagnosis,    ....    29 

DICTIONARIES. 

Dorland — Pocket  Dictionary,     ....  10 

Keating — Pronouncing  Dictionary,    .    .  14 

Morten — Nurse's  Dictionary,     ....  18 


EYE,  EAR,  NOSE,  AND  THROAT. 

An  American  Text- Book  of  Diseases 

of  the  Eye,  Ear,  Nose,  and  Throat,  .  3 
De  Schweinitz — Diseases  of  the  Eye, .  lo 
Gleason — Essentials  of  Dis.  of  the  Ear,  1 1 
Jackson — Manual  of  Diseases  of  Eye,  .  32 
Jackson   and    Gleason — Essentials  of 

Diseases  of  the  Eye,  Nose,  and  Throat,  14 
Kyle — Diseases  of  tlie  Nose  and  Throat,  15 

QENITO=URINARY. 

An  American  Text-Book  of  Genito- 
urinary and  Skin  Diseases, 4 

Hyde  and  Montgomery — Syphilis  and 
the  Venereal  Diseases, ......    13 

Martin — Essentials   of   Minor   Surgery, 

Bandaging,  and  Venereal  Diseases,  .  16 
Saundby — Renal  aiid  Urinary  Diseases,  24 
Senn — Genito- Urinary  Tuberculosis,  .  25 
Vecki — Sexual  Impotence, 28 

GYNECOLOGY. 

American  Text- Book  of  Gynecology, 
Cragin — Essentials  of  Gynecology, 
Garrigues — Diseases  of  Women,  . 
Long — Syllabus  of  Gynecology,  . 
Penrose — Diseases  of  Women,  .  . 
Pryor — Pelvic  Inflammations,  .  . 
Sutton  and  Giles — Diseases  of  Women, 


4 

9 
II 

15 
18 

32 
28 


MATERIA  MEDICA,  PHARMACOL- 
OGY, AND  THERAPEUTICS. 

An  American  Text-Book  of  Applied 

Therapeutics 3 

Butler — Text-Book  of  Materia  Medica, 

Therapeutics  and  Pharmacology,  ...  8 
Cerna — Notes  on  the  Newer  Remedies,  8 
Griffin — Materia  Med.  and  Therapeutics,  12 
Morris — Essentials  of   Materia  Medica 

and  Therapeutics, 17 

Saunders'  Pocket  Medical  Formulary,  24 
Sayre — Essentials  of  Pharmacy,  ...  24 
Stevens — Essentials  of  Materia  Medica 

and  Therapeutics, 26 

Stoney — Materia  Medica  for  Nurses,  .  .  28 
Thornton — Dose-Book  and  Manual  of 

Prescription-Writing, 28 

MEDICAL   JURISPRUDENCE    AND 
TOXICOLOGY. 

Chapman— Medical  Jurisprudence  and 
Toxicology,        ...  ....      8 

Semple — Essentials  of  Legal  Medicine, 
Toxicology,  and  Hygiene, 25 


UNIVERSITY  OF  CALIFORNIA  MEDICAL  SCHOOL  LIBRARY 


